Modified peptides as therapeutic agents

ABSTRACT

The present invention concerns fusion of Fc domains with biologically active peptides and a process for preparing pharmaceutical agents using biologically active peptides. In this invention, pharmacologically active compounds are prepared by a process comprising:
         a) selecting at least one peptide that modulates the activity of a protein of interest; and   b) preparing a pharmacologic agent comprising an Fc domain covalently linked to at least one amino acid of the selected peptide.
 
Linkage to the vehicle increases the half-life of the peptide, which otherwise would be quickly degraded in vivo. The preferred vehicle is an Fc domain. The peptide is preferably selected by phage display,  E. coli  display, ribosome display, RNA-peptide screening, or chemical-peptide screening.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/428,082, filed Oct. 22, 1999 now U.S. Pat. No. 6,660,843, whichclaims the benefit of U.S. Provisional application 60/105,371, filedOct. 23, 1998, which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Recombinant proteins are an emerging class of therapeutic agents. Suchrecombinant therapeutics have engendered advances in protein formulationand chemical modification. Such modifications can protect therapeuticproteins, primarily by blocking their exposure to proteolytic enzymes.Protein modifications may also increase the therapeutic protein'sstability, circulation time, and biological activity. A review articledescribing protein modification and fusion proteins is Francis (1992),Focus on Growth Factors 3:4–10 (Mediscript, London), which is herebyincorporated by reference.

One useful modification is combination with the “Fc” domain of anantibody. Antibodies comprise two functionally independent parts, avariable domain known as “Fab”, which binds antigen, and a constantdomain known as “Fc”, which links to such effector functions ascomplement activation and attack by phagocytic cells. An Fc has a longserum half-life, whereas an Fab is short-lived. Capon et al. (1989),Nature 337: 525–31. When constructed together with a therapeuticprotein, an Fc domain can provide longer half-life or incorporate suchfunctions as Fc receptor binding, protein A binding, complement fixationand perhaps even placental transfer. Id. Table 1 summarizes use of Fcfusions known in the art.

TABLE 1 Fc fusion with therapeutic proteins Fusion Therapeutic Form ofFc partner implications Reference IgG1 N-terminus of Hodgkin's disease;U.S. Pat. No. CD30-L anaplastic lymphoma; T- 5,480,981 cell leukemiaMurine Fcγ2a IL-10 anti-inflammatory; Zheng et al. (1995), J. Immunol.transplant rejection 154: 5590–600 IgG1 TNF receptor septic shock Fisheret al. (1996), N. Engl. J. Med. 334: 1697–1702; Van Zee, K. et al.(1996), J. Immunol. 156: 2221–30 IgG, IgA, TNF receptor inflammation,U.S. Pat. No. 5,808,029, IgM, or IgE autoimmune disorders issued Sep.15, 1998 (excluding the first domain) IgG1 CD4 receptor AIDS Capon etal. (1989), Nature 337: 525–31 IgG1, N-terminus anti-cancer, antiviralHarvill et al. (1995), IgG3 of IL-2 Immunotech. 1: 95–105 IgG1C-terminus of osteoarthritis; WO 97/23614, published OPG bone densityJul. 3, 1997 IgG1 N-terminus of anti-obesity PCT/US 97/23183, filedleptin Dec. 11, 1997 Human Ig CTLA-4 autoimmune disorders Linsley(1991), J. Exp. Cγ1 Med. 174: 561–9

A much different approach to development of therapeutic agents ispeptide library screening. The interaction of a protein ligand with itsreceptor often takes place at a relatively large interface. However, asdemonstrated for human growth hormone and its receptor, only a few keyresidues at the interface contribute to most of the binding energy.Clackson et al. (1995), Science 267: 383–6. The bulk of the proteinligand merely displays the binding epitopes in the right topology orserves functions unrelated to binding. Thus, molecules of only “peptide”length (2 to 40 amino acids) can bind to the receptor protein of a givenlarge protein ligand. Such peptides may mimic the bioactivity of thelarge protein ligand (“peptide agonists”) or, through competitivebinding, inhibit the bioactivity of the large protein ligand (“peptideantagonists”).

Phage display peptide libraries have emerged as a powerful method inidentifying such peptide agonists and antagonists. See, for example,Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No.5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar.12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No.5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul.13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833,published Apr. 16, 1998 (each of which is incorporated by reference). Insuch libraries, random peptide sequences are displayed by fusion withcoat proteins of filamentous phage. Typically, the displayed peptidesare affinity-eluted against an antibody-immobilized extracellular domainof a receptor. The retained phages may be enriched by successive roundsof affinity purification and repropagation. The best binding peptidesmay be sequenced to identify key residues within one or morestructurally related families of peptides. See, e.g., Cwirla et al.(1997), Science 276: 1696–9, in which two distinct families wereidentified. The peptide sequences may also suggest which residues may besafely replaced by alanine scanning or by mutagenesis at the DNA level.Mutagenesis libraries may be created and screened to further optimizethe sequence of the best binders. Lowman (1997), Ann. Rev. Biophys.Biomol. Struct. 26: 401–24.

Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed. See, e.g.,Takasaki et al. (1997), Nature Biotech. 15: 1266–70. These analyticalmethods may also be used to investigate the interaction between areceptor protein and peptides selected by phage display, which maysuggest further modification of the peptides to increase bindingaffinity.

Other methods compete with phage display in peptide research. A peptidelibrary can be fused to the carboxyl terminus of the lac repressor andexpressed in E. coli. Another E. coli-based method allows display on thecell's outer membrane by fusion with a peptidoglycan-associatedlipoprotein (PAL). Hereinafter, these and related methods arecollectively referred to as “E. coli display.” In another method,translation of random RNA is halted prior to ribosome release, resultingin a library of polypeptides with their associated RNA still attached.Hereinafter, this and related methods are collectively referred to as“ribosome display.” Other methods employ chemical linkage of peptides toRNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci.USA, 94:12297–303. Hereinafter, this and related methods arecollectively referred to as “RNA-peptide screening.” Chemically derivedpeptide libraries have been developed in which peptides are immobilizedon stable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.Hereinafter, these and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol.3: 355–62.

Conceptually, one may discover peptide mimetics of any protein usingphage display and the other methods mentioned above. These methods havebeen used for epitope mapping, for identification of critical aminoacids in protein-protein interactions, and as leads for the discovery ofnew therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin.Biotech. 7: 616–21. Peptide libraries are now being used most often inimmunological studies, such as epitope mapping. Kreeger (1996), TheScientist 10(13): 19–20.

Of particular interest here is use of peptide libraries and othertechniques in the discovery of pharmacologically active peptides. Anumber of such peptides identified in the art are summarized in Table 2.The peptides are described in the listed publications, each of which ishereby incorporated by reference. The pharmacologic activity of thepeptides is described, and in many instances is followed by a shorthandterm therefor in parentheses. Some of these peptides have been modified(e.g., to form C-terminally cross-linked dimers). Typically, peptidelibraries were screened for binding to a receptor for apharmacologically active protein (e.g., EPO receptor). In at least oneinstance (CTLA4), the peptide library was screened for binding to amonclonal antibody.

TABLE 2 Pharmacologically active peptides Binding partner/ Form ofprotein of Pharmacologic peptide interest^(a) activity Referenceintrapeptide EPO receptor EPO-mimetic Wrighton et al. (1996), disulfide-Science 273: 458–63; bonded U.S. Pat. No. 5,773,569, issued Jun. 30,1998 to Wrighton et al. C-terminally EPO receptor EPO-mimetic Livnah etal. (1996), cross-linked Science 273: 464–71; dimer Wrighton et al.(1997), Nature Biotechnology 15: 1261–5; International patentapplication WO 96/40772, published Dec. 19, 1996 linear EPO receptorEPO-mimetic Naranda et al. (1999), Proc. Natl. Acad. Sci. USA, 96:7569–74 linear c-Mpl TPO-mimetic Cwirla et al. (1997) Science 276:1696–9; U.S. Pat. No. 5,869,451, issued Feb. 9, 1999; U.S. Pat. No.5,932,946, issued Aug. 3, 1999 C-terminally c-Mpl TPO-mimetic Cwirla etal. (1997), cross-linked Science 276: 1696–9 dimer disulfide-stimulation of Paukovits et al. (1984), linked dimer hematopoiesisHoppe-Seylers Z. (“G-CSF-mimetic”) Physiol. Chem. 365: 303–11; Laerum etal. (1988), Exp. Hemat. 16: 274–80 alkylene- G-CSF-mimetic Bhatnagar etal. (1996), linked dimer J. Med. Chem. 39: 3814–9; Cuthbertson et al.(1997), J. Med. Chem. 40: 2876–82; King et al. (1991), Exp. Hematol. 19:481; King et al. (1995), Blood 86 (Suppl. 1): 309a linear IL-1 receptorinflammatory and U.S Pat. No. 5,608,035; autoimmune diseases U.S. Pat.No. 5,786,331; (“IL-1 antagonist” or U.S. Pat. No. 5,880,096;“IL-1ra-mimetic”) Yanofsky et al. (1996), Proc. Natl. Acad. Sci. 93:7381–6; Akeson et al. (1996), J. Biol. Chem. 271: 30517–23; Wiekzorek etal. (1997), Pol. J. Pharmacol. 49: 107–17; Yanofsky (1996), PNAs, 93:7381–7386. linear Facteur stimulation of Inagaki-Ohara et al. thymiquelymphocytes (1996), Cellular Immunol. serique (FTS) (“FTS-mimetic”) 171:30–40; Yoshida (1984), Int. J. Immunopharmacol, 6: 141–6. intrapeptideCTLA4 MAb CTLA4-mimetic Fukumoto et al. (1998), disulfide NatureBiotech. 16: 267–70 bonded exocyclic TNF-α receptor TNF-α antagonistTakasaki et al. (1997), Nature Biotech. 15: 1266–70; WO 98/53842,published Dec. 3, 1998 linear TNF-α receptor TNF-α antagonistChirinos-Rojas ( ), J. Imm., 5621–5626. intrapeptide C3b inhibition ofcomplement Sahu et al. (1996), J. Immunol. disulfide activation;autoimmune 157: 884–91; bonded diseases Morikis et al. (1998),(“C3b-antagonist”) Protein Sci. 7: 619–27 linear vinculin cell adhesionprocesses - Adey et al. (1997), cell growth, differentiation, Biochem.J. 324: 523–8 wound healing, tumor metastasis (“vinculin binding”)linear C4 binding anti-thrombotic Linse et al. (1997), J. Biol. Chem.protein (C4BP) 272: 14658–65 linear urokinase processes associated withGoodson et al. (1994), receptor urokinase interaction with Proc. Natl.Acad. Sci. 91: its receptor (e.g., 7129–33; International angiogenesis,tumor cell application WO invasion and metastasis); 97/35969, published(“UKR antagonist”) Oct. 2, 1997 linear Mdm2, Hdm2 Inhibition ofinactivation of Picksley et al. (1994), p53 mediated by Mdm2 or Oncogene9: 2523–9; hdm2; anti-tumor Bottger et al. (1997) J. Mol. Biol.(“Mdm/hdm antagonist”) 269: 744–56; Bottger et al. (1996), Oncogene 13:2141–7 linear p21^(WAF1) anti-tumor by mimicking Ball et al. (1997),Curr. the activity of p21^(WAF1) Biol. 7: 71–80 linear farnesylanti-cancer by preventing Gibbs et al. (1994), Cell transferaseactivation of ras oncogene 77: 175–178 linear Ras effector anti-cancerby inhibiting Moodie et al. (1994), domain biological function of theTrends Genet 10: 44–48 ras oncogene Rodriguez et al. (1994), Nature 370:527–532 linear SH2/SH3 anti-cancer by inhibiting Pawson et al (1993),domains tumor growth with Curr. Biol. 3: 434–432 activated tyrosinekinases Yu et al. (1994), Cell 76: 933–945 linear p16^(INK4) anti-cancerby mimicking Fåhraeus et al. (1996), activity of p16; e.g., Curr. Biol.6: 84–91 inhibiting cyclin D-Cdk complex (“p16-mimetic”) linear Src, Lyninhibition of Mast cell Stauffer et al. (1997), activation, IgE-relatedBiochem. 36: 9388–94 conditions, type I hypersensitivity (“Mast cellantagonist”) linear Mast cell treatment of inflammatory Internationalapplication protease disorders mediated by WO 98/33812, publishedrelease of tryptase-6 Aug. 6, 1998 (“Mast cell protease inhibitors”)linear SH3 domains treatment of SH3- Rickles et al. (1994), mediateddisease states EMBO J. 13: 5598–5604; (“SH3 antagonist”) Sparks et al.(1994), J. Biol. Chem. 269: 23853–6; Sparks et al. (1996), Proc. Natl.Acad. Sci. 93: 1540–4 linear HBV core treatment of HBV viral Dyson &Muray (1995), antigen (HBcAg) infections (“anti-HBV”) Proc. Natl. Acad.Sci. 92: 2194–8 linear selectins neutrophil adhesion; Martens et al.(1995), J. Biol. Chem. inflammatory diseases 270: 21129–36; (“selectinantagonist”) European patent application EP 0 714 912, published Jun. 5,1996 linear, calmodulin calmodulin antagonist Pierce et al. (1995),cyclized Molec. Diversity 1: 259–65 Dedman et al. (1993), J. Biol. Chem.268: 23025–30; Adey & Kay (1996), Gene 169: 133–4 linear, integrinstumor-homing; treatment International applications cyclized- forconditions related to WO 95/14714, published integrin-mediated cellularJun. 1, 1995; WO events, including platelet 97/08203, publishedaggregation, thrombosis, Mar. 6, 1997; WO wound healing, 98/10795,published osteoporosis, tissue Mar. 19, 1998; WO repair, angiogenesis(e.g., 99/24462, published May for treatment of cancer), 20, 1999; Kraftet al. and tumor invasion (1999), J. Biol. Chem. (“integrin-binding”)274: 1979–1985 cyclic, linear fibronectin and treatment of inflammatoryWO 98/09985, extracellular and autoimmune published Mar. 12, 1998 matrixconditions components of T cells and macrophages linear somatostatintreatment or prevention of European patent and cortistatinhormone-producing application 0 911 393, tumors, acromegaly, publishedApr. 8, 1999 giantism, dementia, gastric ulcer, tumor growth, inhibitionof hormone secretion, modulation of sleep or neural activity linearbacterial antibiotic; septic shock; U.S. Pat. No. 5,877,151,lipopolysaccharide disorders modulatable by issued Mar. 2, 1999 CAP37linear or pardaxin, antipathogenic WO 97/31019, published cyclic,mellitin Aug. 28, 1997 including D- amino acids linear, cyclic VIPimpotence, WO 97/40070, published neurodegenerative Oct. 30, 1997disorders linear CTLs cancer EP 0 770 624, published May 2, 1997 linearTHF-gamma2 Burnstein (1988), Biochem., 27: 4066–71. linear Amylin Cooper(1987), Proc. Natl. Acad. Sci., 84: 8628–32. linear AdrenomedullinKitamura (1993), BBRC, 192: 553–60. cyclic, linear VEGF anti-angiogenic;cancer, Fairbrother (1998), rheumatoid arthritis, Biochem., 37:17754–17764. diabetic retinopathy, psoriasis (“VEGF antagonist”) cyclicMMP inflammation and Koivunen (1999), Nature autoimmune disorders;Biotech., 17: 768–774. tumor growth (“MMP inhibitor”) HGH fragment U.S.Pat. No. 5,869,452 Echistatin inhibition of platelet Gan (1988), J.Biol. Chem., aggregation 263: 19827–32. linear SLE SLE WO 96/30057,published autoantibody Oct. 3, 1996 GD1alpha suppression of tumorIshikawa et al. (1998), metastasis FEBS Lett. 441 (1): 20–4antiphospholipid endothelial cell activation, Blank et al. (1999), Proc.beta-2- antiphospholipid Natl. Acad. Sci. USA 96: glycoprotein-isyndrome (APS), 5164–8 (β2GPI) thromboembolic antibodies phenomena,thrombocytopenia, and recurrent fetal loss linear T Cell Receptordiabetes WO 96/11214, published beta chain Apr. 18, 1996 ^(a)The proteinlisted in this column may be bound by the associated peptide (e.g., EPOreceptor, IL-1 receptor) or mimicked by the associated peptide. Thereferences listed for each clarify whether the molecule is bound by ormimicked by the peptides. ^(b)FTS is a thymic hormone mimicked by themolecule of this invention rather than a receptor bound by the moleculeof this invention.

Peptides identified by peptide library screening have been regarded as“leads” in development of therapeutic agents rather than as therapeuticagents themselves. Like other proteins and peptides, they would berapidly removed in vivo either by renal filtration, cellular clearancemechanisms in the reticuloendothelial system, or proteolyticdegradation. Francis (1992), Focus on Growth Factors 3: 4–11. As aresult, the art presently uses the identified peptides to validate drugtargets or as scaffolds for design of organic compounds that might nothave been as easily or as quickly identified through chemical libraryscreening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401–24;Kay et al. (1998), Drug Disc. Today 3: 370–8. The art would benefit froma process by which such peptides could more readily yield therapeuticagents.

SUMMARY OF THE INVENTION

The present invention concerns a process by which the in vivo half-lifeof one or more biologically active peptides is increased by fusion witha vehicle. In this invention, pharmacologically active compounds areprepared by a process comprising:

-   -   a) selecting at least one peptide that modulates the activity of        a protein of interest; and    -   b) preparing a pharmacologic agent comprising at least one        vehicle covalently linked to at least one amino acid sequence of        the selected peptide.        The preferred vehicle is an Fc domain. The peptides screened in        step (a) are preferably expressed in a phage display library.        The vehicle and the peptide may be linked through the N- or        C-terminus of the peptide or the vehicle, as described further        below. Derivatives of the above compounds (described below) are        also encompassed by this invention.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Compounds of this invention that encompassnon-peptide portions may be synthesized by standard organic chemistryreactions, in addition to standard peptide chemistry reactions whenapplicable.

The primary use contemplated is as therapeutic or prophylactic agents.The vehicle-linked peptide may have activity comparable to—or evengreater than—the natural ligand mimicked by the peptide. In addition,certain natural ligand-based therapeutic agents might induce antibodiesagainst the patient's own endogenous ligand; the vehicle-linked peptideavoids this pitfall by having little or typically no sequence identitywith the natural ligand.

Although mostly contemplated as therapeutic agents, compounds of thisinvention may also be useful in screening for such agents. For example,one could use an Fc-peptide (e.g., Fc-SH2 domain peptide) in an assayemploying anti-Fc coated plates. The vehicle, especially Fc, may makeinsoluble peptides soluble and thus useful in a number of assays.

The compounds of this invention may be used for therapeutic orprophylactic purposes by formulating them with appropriatepharmaceutical carrier materials and administering an effective amountto a patient, such as a human (or other mammal) in need thereof. Otherrelated aspects are also included in the instant invention.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of an exemplary process of theinvention. In this preferred process, the vehicle is an Fc domain, whichis linked to the peptide covalently by expression from a DNA constructencoding both the Fc domain and the peptide. As noted in FIG. 1, the Fcdomains spontaneously form a dimer in this process.

FIG. 2 shows exemplary Fc dimers that may be derived from an IgG1antibody. “Fc” in the figure represents any of the Fc variants withinthe meaning of “Fc domain” herein. “X¹” and “X²” represent peptides orlinker-peptide combinations as defined hereinafter. The specific dimersare as follows:

A, D: Single disulfide-bonded dimers. IgG1 antibodies typically have twodisulfide bonds at the hinge region between the constant and variabledomains. The Fc domain in FIGS. 2A and 2D may be formed by truncationbetween the two disulfide bond sites or by substitution of a cysteinylresidue with an unreactive residue (e.g., alanyl). In FIG. 2A, the Fcdomain is linked at the amino terminus of the peptides; in 2D, at thecarboxyl terminus.

B, E: Doubly disulfide-bonded dimers. This Fc domain may be formed bytruncation of the parent antibody to retain both cysteinyl residues inthe Fc domain chains or by expression from a construct including asequence encoding such an Fc domain. In FIG. 2B, the Fc domain is linkedat the amino terminus of the peptides; in 2E, at the carboxyl terminus.

C, F: Noncovalent dimers. This Fc domain may be formed by elimination ofthe cysteinyl residues by either truncation or substitution. One maydesire to eliminate the cysteinyl residues to avoid impurities formed byreaction of the cysteinyl residue with cysteinyl residues of otherproteins present in the host cell. The noncovalent bonding of the Fcdomains is sufficient to hold together the dimer.

Other dimers may be formed by using Fc domains derived from differenttypes of antibodies (e.g., IgG2, IgM).

FIG. 3 shows the structure of preferred compounds of the invention thatfeature tandem repeats of the pharmacologically active peptide. FIG. 3Ashows a single chain molecule and may also represent the DNA constructfor the molecule. FIG. 3B shows a dimer in which the linker-peptideportion is present on only one chain of the dimer. FIG. 3C shows a dimerhaving the peptide portion on both chains. The dimer of FIG. 3C willform spontaneously in certain host cells upon expression of a DNAconstruct encoding the single chain shown in FIG. 3A. In other hostcells, the cells could be placed in conditions favoring formation ofdimers or the dimers can be formed in vitro.

FIG. 4 shows exemplary nucleic acid and amino acid sequences (SEQ IDNOS: 1 and 2, respectively) of human IgG1 Fc that may be used in thisinvention.

FIG. 5 shows a synthetic scheme for the preparation of PEGylated peptide19 (SEQ ID NO: 3) as prepared through intermediates having SEQ ID NOS:1128 through 1131, respectively.

FIG. 6 shows a synthetic scheme for the preparation of PEGylated peptide20 (SEQ ID NO: 4) as prepared through intermediates having SEQ ID NOS:1132 and 1133, respectively.

FIG. 7 shows the nucleotide and amino acid sequences (SEQ ID NOS: 5 and6, respectively) of the molecule identified as “Fc-TMP” in Example 2hereinafter.

FIG. 8 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 7and 8, respectively) of the molecule identified as “Fc-TMP-TMP” inExample 2 hereinafter.

FIG. 9 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 9and 10, respectively) of the molecule identified as “TMP-TMP-Fc” inExample 2 hereinafter.

FIG. 10 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 11and 12, respectively) of the molecule identified as “TMP-Fc” in Example2 hereinafter.

FIG. 11 shows the number of platelets generated in vivo in normal femaleBDF1 mice treated with one 100 μg/kg bolus injection of variouscompounds, with the terms defined as follows.

-   -   PEG-MGDF: 20 kD average molecular weight PEG attached by        reductive amination to the N-terminal amino group of amino acids        1–163 of native human TPO, which is expressed in E. coli (so        that it is not glycosylated);    -   TMP: the TPO-mimetic peptide having the amino acid sequence        IEGPTLRQWLAARA (SEQ ID NO: 13);    -   TMP-TMP: the TPO-mimetic peptide having the amino acid sequence        IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ ID NO: 14);    -   PEG-TMP-TMP: the peptide of SEQ ID NO: 14, wherein the PEG group        is a 5 kD average molecular weight PEG attached as shown in FIG.        6;    -   Fc-TMP-TMP: the compound of SEQ ID NO: 8 (FIG. 8) dimerized with        an identical second monomer (i.e., Cys residues 7 and 10 are        bound to the corresponding Cys residues in the second monomer to        form a dimer, as shown in FIG. 2); and    -   TMP-TMP-Fc is the compound of SEQ ID NO: 10 (FIG. 9) dimerized        in the same way as TMP-TMP-Fc except that the Fc domain is        attached at the C-terminal end rather than the N-terminal end of        the TMP-TMP peptide.

FIG. 12 shows the number of platelets generated in vivo in normal BDF1mice treated with various compounds delivered via implanted osmoticpumps over a 7-day period. The compounds are as defined for FIG. 7.

FIG. 13 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 15and 16, respectively) of the molecule identified as “Fc-EMP” in Example3 hereinafter.

FIG. 14 shows the nucleotide and amino acid sequences (SEQ ID NOS: 17and 18, respectively) of the molecule identified as “EMP-Fc” in Example3 hereinafter.

FIG. 15 shows the nucleotide and amino acid sequences (SEQ ID NOS:19 and20, respectively) of the molecule identified as “EMP-EMP-Fc” in Example3 hereinafter.

FIG. 16 shows the nucleotide and amino acid sequences (SEQ ID NOS: 21and 22, respectively) of the molecule identified as “Fc-EMP-EMP” inExample 3 hereinafter.

FIGS. 17A and 17B show the DNA sequence (SEQ ID NO: 23) inserted intopCFM1656 between the unique AatII (position #4364 in pCFM1656) and SacII(position #4585 in pCFM1656) restriction sites to form expressionplasmid pAMG21 (ATCC accession no. 98113).

FIG. 18A shows the hemoglobin, red blood cells, and hematocrit generatedin vivo in normal female BDF1 mice treated with one 100 μg/kg bolusinjection of various compounds. FIG. 18B shows the same results withmice treated with 100 μg/kg per day delivered the same dose by 7-daymicro-osmotic pump with the EMPs delivered at 100 μg/kg, rhEPO at 30U/mouse. (In both experiments, neutrophils, lymphocytes, and plateletswere unaffected.) In these figures, the terms are defined as follows.

-   -   Fc-EMP: the compound of SEQ ID NO: 16 (FIG. 13) dimerized with        an identical second monomer (i.e., Cys residues 7 and 10 are        bound to the corresponding Cys residues in the second monomer to        form a dimer, as shown in FIG. 2);    -   EMP-Fc: the compound of SEQ ID NO: 18 (FIG. 14) dimerized in the        same way as Fc-EMP except that the Fc domain is attached at the        C-terminal end rather than the N-terminal end of the EMP        peptide.    -   “EMP-EMP-Fc” refers to a tandem repeat of the same peptide (SEQ        ID NO: 20) attached to the same Fc domain by the carboxyl        terminus of the peptides. “Fc-EMP-EMP” refers to the same tandem        repeat of the peptide but with the same Fc domain attached at        the amino terminus of the tandem repeat. All molecules are        expressed in E. coli and so are not glycosylated.

FIGS. 19A and 19B show the nucleotide and amino acid sequences (SEQ IDNOS: 1055 and 1056) of the Fc-TNF-α inhibitor fusion molecule describedin Example 4 hereinafter.

FIGS. 20A and 20B show the nucleotide and amino acid sequences (SEQ IDNOS: 1057 and 1058) of the TNF-α inhibitor-Fc fusion molecule describedin Example 4 hereinafter.

FIGS. 21A and 21B show the nucleotide and amino acid sequences (SEQ IDNOS: 1059 and 1060) of the Fc-IL-1 antagonist fusion molecule describedin Example 5 hereinafter.

FIGS. 22A and 22B show the nucleotide and amino acid sequences (SEQ IDNOS: 1061 and 1062) of the IL-1 antagonist-Fc fusion molecule describedin Example 5 hereinafter.

FIGS. 23A and 23B show the nucleotide and amino acid sequences (SEQ IDNOS: 1063 and 1064) of the Fc-VEGF antagonist fusion molecule describedin Example 6 hereinafter.

FIGS. 24A and 24B show the nucleotide and amino acid sequences (SEQ IDNOS: 1065 and 1066) of the VEGF antagonist-Fc fusion molecule describedin Example 6 hereinafter.

FIGS. 25A and 25B show the nucleotide and amino acid sequences (SEQ IDNOS: 1067 and 1068) of the Fc-MMP inhibitor fusion molecule described inExample 7 hereinafter.

FIGS. 26A and 26B show the nucleotide and amino acid sequences (SEQ IDNOS: 1069 and 1070) of the MMP inhibitor-Fc fusion molecule described inExample 7 hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances.

The term “comprising” means that a compound may include additional aminoacids on either or both of the N- or C-termini of the given sequence. Ofcourse, these additional amino acids should not significantly interferewith the activity of the compound.

The term “vehicle” refers to a molecule that prevents degradation and/orincreases half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of a therapeutic protein. Exemplaryvehicles include an Fc domain (which is preferred) as well as a linearpolymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); abranched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 toDenkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 toTam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28Oct. 1993); a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide; or any natural or synthetic protein,polypeptide or peptide that binds to a salvage receptor. Vehicles arefurther described hereinafter.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071–9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. International applications WO 97/34631 (published Sep.25, 1997) and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. Thus, the term “Fc variant” comprises a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises sites that may be removed because they provide structuralfeatures or biological activity that are not required for the fusionmolecules of the present invention. Thus, the term “Fc variant”comprises a molecule or sequence that lacks one or more native Fc sitesor residues that affect or are involved in (1) disulfide bond formation,(2) incompatibility with a selected host cell (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Thus, exemplary dimers within the scope ofthis invention are as shown in FIG. 2.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 2 to 40 amino acids, withmolecules of 3 to 20 amino acids preferred and those of 6 to 15 aminoacids most preferred. Exemplary peptides may be randomly generated byany of the methods cited above, carried in a peptide library (e.g., aphage display library), or derived by digestion of proteins.

The term “randomized” as used to refer to peptide sequences refers tofully random sequences (e.g., selected by phage display methods) andsequences in which one or more residues of a naturally occurringmolecule is replaced by an amino acid residue not appearing in thatposition in the naturally occurring molecule. Exemplary methods foridentifying peptide sequences include phage display, E. coli display,ribosome display, RNA-peptide screening, chemical screening, and thelike.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer, autoimmune disorders). Thus, pharmacologically activepeptides comprise agonistic or mimetic and antagonistic peptides asdefined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptidehaving biological activity comparable to a protein (e.g., EPO, TPO,G-CSF) that interacts with a protein of interest. These terms furtherinclude peptides that indirectly mimic the activity of a protein ofinterest, such as by potentiating the effects of the natural ligand ofthe protein of interest; see, for example, the G-CSF-mimetic peptideslisted in Tables 2 and 7. Thus, the term “EPO-mimetic peptide” comprisesany peptides that can be identified or derived as described in Wrightonet al. (1996), Science 273: 458–63, Naranda et al. (1999), Proc. Natl.Acad. Sci. USA 96: 7569–74, or any other reference in Table 2 identifiedas having EPO-mimetic subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “TPO-mimetic peptide” comprises peptides that can be identifiedor derived as described in Cwirla et al. (1997), Science 276: 1696–9,and hereby incorporated by reference. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “G-CSF-mimetic peptide” comprises any peptides that can beidentified or described in Paukovits et al. (1984), Hoppe-Seylers Z.Physiol. Chem. 365: 303–11 or any of the references in Table 2identified as having G-CSF-mimetic subject matter. Those of ordinaryskill in the art appreciate that each of these references enables one toselect different peptides than actually disclosed therein by followingthe disclosed procedures with different peptide libraries.

The term “CTLA4-mimetic peptide” comprises any peptides that can beidentified or derived as described in Fukumoto et al. (1998), NatureBiotech. 16: 267–70. Those of ordinary skill in the art appreciate thateach of these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

The term “-antagonist peptide” or “inhibitor peptide” refers to apeptide that blocks or in some way interferes with the biologicalactivity of the associated protein of interest, or has biologicalactivity comparable to a known antagonist or inhibitor of the associatedprotein of interest. Thus, the term “TNF-antagonist peptide” comprisespeptides that can be identified or derived as described in Takasaki etal. (1997), Nature Biotech. 15: 1266–70 or any of the references inTable 2 identified as having TNF-antagonistic subject matter. Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The terms “IL-1 antagonist” and “IL-1ra-mimetic peptide” comprisespeptides that inhibit or down-regulate activation of the IL-1 receptorby IL-1. IL-1 receptor activation results from formation of a complexamong IL-1, IL-1 receptor, and IL-1 receptor accessory protein. IL-1antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor, orIL-1 receptor accessory protein and obstruct complex formation among anytwo or three components of the complex. Exemplary IL-1 antagonist orIL-1ra-mimetic peptides can be identified or derived as described inU.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096, or any of the referencesin Table 2 identified as having IL-1ra-mimetic or IL-1 antagonisticsubject matter. Those of ordinary skill in the art appreciate that eachof these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

The term “VEGF-antagonist peptide” comprises peptides that can beidentified or derived as described in Fairbrother (1998), Biochem. 37:17754–64, and in any of the references in Table 2 identified as havingVEGF-antagonistic subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “MMP inhibitor peptide” comprises peptides that can beidentified or derived as described in Koivunen (1999), Nature Biotech.17: 768–74 and in any of the references in Table 2 identified as havingMMP inhibitory subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

Additionally, physiologically acceptable salts of the compounds of thisinvention are also encompassed herein. By “physiologically acceptablesalts” is meant any salts that are known or later discovered to bepharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate.

Structure of Compounds

In General. In the compositions of matter prepared in accordance withthis invention, the peptide may be attached to the vehicle through thepeptide's N-terminus or C-terminus. Thus, the vehicle-peptide moleculesof this invention may be described by the following formula I:(X¹)_(a)—F¹—(X²)_(b)  Iwherein:

F¹ is a vehicle (preferably an Fc domain);

X¹ and X² are each independently selected from -(L¹)_(c)-P¹,-(L¹)_(c)-P¹-(L²)_(d)-P², -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴

P¹, P², P³, and P⁴ are each independently sequences of pharmacologicallyactive peptides;

L¹, L², L³, and L⁴ are each independently linkers; and

a, b, c, d, e, and f are each independently 0 or 1, provided that atleast one of a and b is 1.

Thus, compound I comprises preferred compounds of the formulaeX¹—F¹  IIand multimers thereof wherein F¹ is an Fc domain and is attached at theC-terminus of X¹;F¹—X²  IIIand multimers thereof wherein F¹ is an Fc domain and is attached at theN-terminus of X²;F¹-(L¹)_(c)-P¹  IVand multimers thereof wherein F¹ is an Fc domain and is attached at theN-terminus of -(L¹)_(c)-P¹; andF¹-(L¹)_(c)-P¹-(L²)_(d)-P²  Vand multimers thereof wherein F¹ is an Fc domain and is attached at theN-terminus of -L¹-P¹-L²-P².

Peptides. Any number of peptides may be used in conjunction with thepresent invention. Of particular interest are peptides that mimic theactivity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, leptin,CTLA4, TRAIL, TGF-α, and TGF-β. Peptide antagonists are also ofinterest, particularly those antagonistic to the activity of TNF,leptin, any of the interleukins (IL-1, 2, 3, . . . ), and proteinsinvolved in complement activation (e.g., C3b). Targeting peptides arealso of interest, including tumor-homing peptides, membrane-transportingpeptides, and the like. All of these classes of peptides may bediscovered by methods described in the references cited in thisspecification and other references.

Phage display, in particular, is useful in generating peptides for usein the present invention. It has been stated that affinity selectionfrom libraries of random peptides can be used to identify peptideligands for any site of any gene product. Dedman et al. (1993), J. Biol.Chem. 268: 23025–30. Phage display is particularly well suited foridentifying peptides that bind to such proteins of interest as cellsurface receptors or any proteins having linear epitopes. Wilson et al.(1998), Can. J. Microbiol. 44: 313–29; Kay et al. (1998), Drug Disc.Today 3: 370–8. Such proteins are extensively reviewed in Herz et al.(1997), J. Receptor & Signal Transduction Res. 17(5): 671–776, which ishereby incorporated by reference. Such proteins of interest arepreferred for use in this invention.

A particularly preferred group of peptides are those that bind tocytokine receptors. Cytokines have recently been classified according totheir receptor code. See Inglot (1997), Archivum Immunologiae etTherapiae Experimentalis 45: 353–7, which is hereby incorporated byreference. Among these receptors, most preferred are the CKRs (family Iin Table 3). The receptor classification appears in Table 3.

TABLE 3 Cytokine Receptors Classified by Receptor Code Cytokines(ligands) Receptor Type family subfamily family subfamily I.Hematopoietic 1. IL-2, IL-4, IL-7, I. Cytokine R (CKR) 1. shared γCr  cytokines   IL-9, IL-13, IL-   15 2. IL-3, IL-5, GM- 2. shared GP 140  CSF   βR 3. IL-6, IL-11, IL- 3. 3.shared RP   12, LIF, OSM,   130  CNTF, leptin   (OB) 4. G-CSF, EPO, 4. “single chain” R   TPO, PRL, GH5. IL-17, HVS-IL- 5. other R^(c)   17 II. IL-10 ligands IL-10, BCRF-1,II. IL-10 R HSV-IL-10 III. Interferons 1. IFN-α1, α2, α4, III.Interferon R 1. IFNAR   m, t, IFN-β^(d) 2. IFN-γ 2. IFNGR IV. IL-1ligands 1. IL-1α, IL-1β, IL- IV. IL-1R   1Ra V. TNF ligands 1. TNF-α,TNF-β V. NGF/TNF R^(e)   (LT), FAS1,   CD40 L,   CD30L, CD27 L VI.Chemokines 1. α chemokines: VI. Chemokine R 1. CXCR   IL-8, GRO   α, β,γ, IF-10,   PF-4, SDF-1 2. β chemokines: 2. CCR   MIP1α, MIP1β,   MCP-1,2, 3, 4,   RANTES,   eotaxin 3. γ chemokines: 3. CR   lymphotactin 4.DARC^(f) VII. Growth factors 1.1 SCF, VII. RKF 1. TK sub-family   M-CSF, 1.1 IgTK III R    PDGF-AA, AB,    BB, FLT-3L,    VEGF, SSV-   PDGF 1.2 FGFα, FGFβ 1.2 IgTK IV R 1.3 EGF, TGF-α, 1.3 Cysteine-richTK-I    VV-F19 (EGF-    like) 1.4 IGF-I, IGF-II, 1.4 Cysteine rich TK-II   Insulin 1.5 NGF, BDNF, 1.5 Cysteine knot TK V    NT-3, NT-4^(g) 2.TGF-β1, β2, β3 2. STK subfamily^(h) ^(c)IL-17R belongs to the CKR familybut is not assigned to any of the 4 indicated subjamilies. ^(d)Other IFNtype I subtypes remain unassigned. Hematopoietic cytokines, IL-10ligands and interferons do not possess functional intrinsic proteinkinases. The signaling molecules for the cytokines are JAK's, STATs andrelated non-receptor molecules. IL-14, IL-16 and IL-18 have been clonedbut according to the receptor code they remain unassigned. ^(e)TNFreceptors use multiple, distinct intracellular molecules for signaltransduction including “death domain” of FAS R and 55 kDa TNF-αR thatparticipates in their cytotoxic effects. NGF/TNF R can bind both NGF andrelated factors as well as TNF ligands. Chemokine receptors are Gprotein-coupled, seven transmembrane (7TM, serpentine) domain receptors.^(f)The Duffy blood group antigen (DARC) is an erythrocyte receptor thatcan bind several different chemokines. It belongs to the immunoglobulinsuperfamily but characteristics of its signal transduction events remainunclear. ^(g)The neurotrophic cytokines can associate with NGF/TNFreceptors also. ^(h)STKS may encompass many other TGF-β-related factorsthat remain unassigned. The protein kinases are intrinsic part of theintracellular domain of receptor kinase family (RKF). The enzymesparticipate in the signals transmission via the receptors.

Exemplary peptides for this invention appear in Tables 4 through 20below. These peptides may be prepared by methods disclosed in the art.Single letter amino acid abbreviations are used. The X in thesesequences (and throughout this specification, unless specified otherwisein a particular instance) means that any of the 20 naturally occurringamino acid residues may be present. Any of these peptides may be linkedin tandem (i.e., sequentially), with or without linkers, and a fewtandem-linked examples are provided in the table. Linkers are listed as“Λ” and may be any of the linkers described herein. Tandem repeats andlinkers are shown separated by dashes for clarity. Any peptidecontaining a cysteinyl residue may be cross-linked with anotherCys-containing peptide, either or both of which may be linked to avehicle. A few cross-linked examples are provided in the table. Anypeptide having more than one Cys residue may form an intrapeptidedisulfide bond, as well; see, for example, EPO-mimetic peptides in Table5. A few examples of intrapeptide disulfide-bonded peptides arespecified in the table. Any of these peptides may be derivatized asdescribed herein, and a few derivatized examples are provided in thetable. Derivatized peptides in the tables are exemplary rather thanlimiting, as the associated underivatized peptides may be employed inthis invention, as well. For derivatives in which the carboxyl terminusmay be capped with an amino group, the capping amino group is shown as—NH₂. For derivatives in which amino acid residues are substituted bymoieties other than amino acid residues, the substitutions are denotedby σ, which signifies any of the moieties described in Bhatnagar et al.(1996), J. Med. Chem. 39: 3814–9 and Cuthbertson et al. (1997), J. Med.Chem. 40: 2876–82, which are incorporated by reference. The Jsubstituent and the Z substituents (Z₅, Z₆, . . . Z₄₀) are as defined inU.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which areincorporated by reference. For the EPO-mimetic sequences (Table 5), thesubstituents X₂ through X₁₁ and the integer “n” are as defined in WO96/40772, which is incorporated by reference. The substituents “Ψ,” “Θ,”and “+” are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci.93: 1540–4, which is hereby incorporated by reference. X₄, X₅, X₆, andX₇ are as defined in U.S. Pat. No. 5,773,569, which is herebyincorporated by reference, except that: for integrin-binding peptides,X₁, X₂, X₃, X₄, X₅, X₆, X₇, and X₈ are as defined in Internationalapplications WO 95/14714, published Jun. 1, 1995 and WO 97/08203,published Mar. 6, 1997, which are also incorporated by reference; andfor VIP-mimetic peptides, X₁, X₁′, X₁″, X₂, X₃, X₄, X₅, X₆ and Z and theintegers m and n are as defined in WO 97/40070, published Oct. 30, 1997,which is also incorporated by reference. Xaa and Yaa below are asdefined in WO 98/09985, published Mar. 12, 1998, which is incorporatedby reference. AA₁, AA₂, AB₁, AB₂, and AC are as defined in Internationalapplication WO 98/53842, published Dec. 3, 1998, which is incorporatedby reference. X¹, X², X³, and X⁴ in Table 17 only are as defined inEuropean application EP 0 911 393, published Apr. 28, 1999. Residuesappearing in boldface are D-amino acids. All peptides are linked throughpeptide bonds unless otherwise noted. Abbreviations are listed at theend of this specification. In the “SEQ ID NO.” column, “NR” means thatno sequence listing is required for the given sequence.

TABLE 4 IL-1 antagonist peptide sequences Sequence/structure SEQ ID NO:Z₁₁Z₇Z₈QZ₅YZ₆Z₉Z₁₀ 212 XXQZ₅YZ₆XX 907 Z₇XQZ₅YZ₆XX 908 Z₇Z₈QZ₅YZ₆Z₉Z₁₀909 Z₁₁Z₇Z₈QZ₅YZ₆Z₉Z₁₀ 910Z₁₂Z₁₃Z₁₄Z₁₅Z₁₆Z₁₇Z₁₈Z₁₉Z₂₀Z₂₁Z₂₂Z₁₁Z₇Z₈QZ₅YZ₆Z₉Z₁₀L 917Z₂₃NZ₂₄Z₃₉Z₂₅Z₂₆Z₂₇Z₂₈Z₂₉Z₃₀Z₄₀ 979 TANVSSFEWTPYYWQPYALPL 213SWTDYGYWQPYALPISGL 214 ETPFTWEESNAYYWQPYALPL 215 ENTYSPNWADSMYWQPYALPL216 SVGEDHNFWTSEYWQPYALPL 217 DGYDRWRQSGERYWQPYALPL 218 FEWTPGYWQPY 219FEWTPGYWQHY 220 FEWTPGWYQJY 221 AcFEWTPGWYQJY 222 FEWTPGWpYQJY 223FAWTPGYWQJY 224 FEWAPGYWQJY 225 FEWVPGYWQJY 226 FEWTPGYWQJY 227AcFEWTPGYWQJY 228 FEWTPaWYQJY 229 FEWTPSarWYQJY 230 FEWTPGYYQPY 231FEWTPGWWQPY 232 FEWTPNYWQPY 233 FEWTPvYWQJY 234 FEWTPecGYWQJY 235FEWTPAibYWQJY 236 FEWTSarGYWQJY 237 FEWTPGYWQPY 238 FEWTPGYWQHY 239FEWTPGWYQJY 240 AcFEWTPGWYQJY 241 FEWTPGW-pY-QJY 242 FAWTPGYWQJY 243FEWAPGYWQJY 244 FEWVPGYWQJY 245 FEWTPGYWQJY 246 AcFEWTPGYWQJY 247FEWTPAWYQJY 248 FEWTPSarWYQJY 249 FEWTPGYYQPY 250 FEWTPGWWQPY 251FEWTPNYWQPY 252 FEWTPVYWQJY 253 FEWTPecGYWQJY 254 FEWTPAibYWQJY 255FEWTSarGYWQJY 256 FEWTPGYWQPYALPL 257 1NapEWTPGYYQJY 258 YEWTPGYYQJY 259FEWVPGYYQJY 260 FEWTPSYYQJY 261 FEWTPNYYQJY 262 TKPR 263 RKSSK 264 RKQDK265 NRKQDK 266 RKQDKR 267 ENRKQDKRF 268 VTKFYF 269 VTKFY 270 VTDFY 271SHLYWQPYSVQ 671 TLVYWQPYSLQT 672 RGDYWQPYSVQS 673 VHVYWQPYSVQT 674RLVYWQPYSVQT 675 SRVWFQPYSLQS 676 NMVYWQPYSIQT 677 SVVFWQPYSVQT 678TFVYWQPYALPL 679 TLVYWQPYSIQR 680 RLVYWQPYSVQR 681 SPVFWQPYSIQI 682WIEWWQPYSVQS 683 SLIYWQPYSLQM 684 TRLYWQPYSVQR 685 RCDYWQPYSVQT 686MRVFWQPYSVQN 687 KIVYWQPYSVQT 688 RHLYWQPYSVQR 689 ALVWWQPYSEQI 690SRVWFQPYSLQS 691 WEQPYALPLE 692 QLVWWQPYSVQR 693 DLRYWQPYSVQV 694ELVWWQPYSLQL 695 DLVWWQPYSVQW 696 NGNYWQPYSFQV 697 ELVYWQPYSIQR 698ELMYWQPYSVQE 699 NLLYWQPYSMQD 700 GYEWYQPYSVQR 701 SRVWYQPYSVQR 702LSEQYQPYSVQR 703 GGGWWQPYSVQR 704 VGRWYQPYSVQR 705 VHVYWQPYSVQR 706QARWYQPYSVQR 707 VHVYWQPYSVQT 708 RSVYWQPYSVQR 709 TRVWFQPYSVQR 710GRIWFQPYSVQR 711 GRVWFQPYSVQR 712 ARTWYQPYSVQR 713 ARVWWQPYSVQM 714RLMFYQPYSVQR 715 ESMWYQPYSVQR 716 HFGWWQPYSVHM 717 ARFWWQPYSVQR 718RLVYWQ PYAPIY 719 RLVYWQ PYSYQT 720 RLVYWQ PYSLPI 721 RLVYWQ PYSVQA 722SRVWYQ PYAKGL 723 SRVWYQ PYAQGL 724 SRVWYQ PYAMPL 725 SRVWYQ PYSVQA 726SRVWYQ PYSLGL 727 SRVWYQ PYAREL 728 SRVWYQ PYSRQP 729 SRVWYQ PYFVQP 730EYEWYQ PYALPL 731 IPEYWQ PYALPL 732 SRIWWQ PYALPL 733 DPLFWQ PYALPL 734SRQWVQ PYALPL 735 IRSWWQ PYALPL 736 RGYWQ PYALPL 737 RLLWVQ PYALPL 738EYRWFQ PYALPL 739 DAYWVQ PYALPL 740 WSGYFQ PYALPL 741 NIEFWQ PYALPL 742TRDWVQ PYALPL 743 DSSWYQ PYALPL 744 IGNWYQ PYALPL 745 NLRWDQ PYALPL 746LPEFWQ PYALPL 747 DSYWWQ PYALPL 748 RSQYYQ PYALPL 749 ARFWLQ PYALPL 750NSYFWQ PYALPL 751 RFMYWQPYSVQR 752 AHLFWQPYSVQR 753 WWQPYALPL 754YYQPYALPL 755 YFQPYALGL 756 YWYQPYALPL 757 RWWQPYATPL 758 GWYQPYALGF 759YWYQPYALGL 760 IWYQPYAMPL 761 SNMQPYQRLS 762 TEVYWQPY AVGLPAAETACN 763TFVYWQPY SVQMTITGKVTM 764 TEVYWQPY SSHXXVPXGFPL 765 TFVYWQPYYGNPQWAIHVRH 766 TFVYWQPY VLLELPEGAVRA 767 TFVYWQPY VDYVWPIPIAQV 768GWYQPYVDGWR 769 RWEQPYVKDGWS 770 EWYQPYALGWAR 771 GWWQPYARGL 772LFEQPYAKALGL 773 GWEQPYARGLAG 774 AWVQPYATPLDE 775 MWYQPYSSQPAE 776GWTQPYSQQGEV 777 DWFQPYSIQSDE 778 PWIQPYARGFG 779 RPLYWQPYSVQV 780TLIYWQPYSVQI 781 RFDYWQPYSDQT 782 WHQFVQPYALPL 783 EWDS VYWQPYSVQ TLLR784 WEQN VYWQPYSVQ SFAD 785 SDV VYWQPYSVQ SLEM 786 YYDG VYWQPYSVQ VMPA787 SDIWYQ PYALPL 788 QRIWWQ PYALPL 789 SRIWWQ PYALPL 790 RSLYWQ PYALPL791 TIIWEQ PYALPL 792 WETWYQ PYALPL 793 SYDWEQ PYALPL 794 SRIWCQ PYALPL795 EIMFWQ PYALPL 796 DYVWQQ PYALPL 797 MDLLVQ WYQPYALPL 798 GSKVILWYQPYALPL 799 RQGANI WYQPYALPL 800 GGGDEP WYQPYALPL 801 SQLERT WYQPYALPL802 ETWVRE WYQPYALPL 803 KKGSTQ WYQPYALPL 804 LQARMN WYQPYALPL 805EPRSQK WYQPYALPL 806 VKQKWR WYQPYALPL 807 LRRHDV WYQPYALPL 808 RSTASIWYQPYALPL 809 ESKEDQ WYQPYALPL 810 EGLTMK WYQPYALPL 811 EGSREG WYQPYALPL812 VIEWWQ PYALPL 813 VWYWEQ PYALPL 814 ASEWWQ PYALPL 815 FYEWWQ PYALPL816 EGWWVQ PYALPL 817 WGEWLQ PYALPL 818 DYVWEQ PYALPL 819 AHTWWQ PYALPL820 FIEWFQ PYALPL 821 WLAWEQ PYALPL 822 VMEWWQ PYALPL 823 ERMWQ PYALPL824 NXXWXX PYALPL 825 WGNWYQ PYALPL 826 TLYWEQ PYALPL 827 VWRWEQ PYALPL828 LLWTQ PYALPL 829 SRIWXX PYALPL 830 SDIWYQ PYALPL 831 WGYYXX PYALPL832 TSGWYQ PYALPL 833 VHPYXX PYALPL 834 EHSYFQ PYALPL 835 XXIWYQ PYALPL836 AQLHSQ PYALPL 837 WANWFQ PYALPL 838 SRLYSQ PYALPL 839 GVTFSQ PYALPL840 SIVWSQ PYALPL 841 SRDLVQ PYALPL 842 HWGH VYWQPYSVQ DDLG 843 SWHSVYWQPYSVQ SVPE 844 WRDS VYWQPYSVQ PESA 845 TWDA VYWQPYSVQ KWLD 846 TPPWVYWQPYSVQ SLDP 847 YWSS VYWQPYSVQ SVHS 848 YWY QPY ALGL 849 YWY QPY ALPL850 EWI QPY ATGL 851 NWE QPY AKPL 852 AFY QPY ALPL 853 FLY QPY ALPL 854VCK QPY LEWC 855 ETPFTWEESNAYYWQPYALPL 856 QGWLTWQDSVDMYWQPYALPL 857FSEAGYTWPENTYWQPYALPL 858 TESPGGLDWAKIYWQPYALPL 859DGYDRWRQSGERYWQPYALPL 860 TANVSSFEWTPGYWQPYALPL 861 SVGEDHNFWTSEYWQPYALPL 862 MNDQTSEVSTFP YWQPYALPL 863 SWSEAFEQPRNL YWQPYALPL 864QYAEPSALNDWG YWQPYALPL 865 NGDWATADWSNY YWQPYALPL 866 THDEHI YWQPYALPL867 MLEKTYTTWTPG YWQPYALPL 868 WSDPLTRDADL YWQPYALPL 869 SDAFTTQDSQAMYWQPYALPL 870 GDDAAWRTDSLT YWQPYALPL 871 AIIRQLYRWSEM YWQPYALPL 872ENTYSPNWADSM YWQPYALPL 873 MNDQTSEVSTFP YWQPYALPL 874 SVGEDHNFWTSEYWQPYALPL 875 QTPFTWEESNAY YWQPYALPL 876 ENPFTWQESNAY YWQPYALPL 877VTPFTWEDSNVF YWQPYALPL 878 QIPFTWEQSNAY YWQPYALPL 879 QAPLTWQESAAYYWQPYALPL 880 EPTFTWEESKAT YWQPYALPL 881 TTTLTWEESNAY YWQPYALPL 882ESPLTWEESSAL YWQPYALPL 883 ETPLTWEESNAY YWQPYALPL 884 EATFTWAESNAYYWQPYALPL 885 EALFTWKESTAY YWQPYALPL 886 STP-TWEESNAY YWQPYALPL 887ETPFTWEESNAY YWQPYALPL 888 KAPFTWEESQAY YWQPYALPL 889 STSFTWEESNAYYWQPYALPL 890 DSTFTWEESNAY YWQPYALPL 891 YIPFTWEESNAY YWQPYALPL 892QTAFTWEESNAY YWQPYALPL 893 ETLFTWEESNAT YWQPYALPL 894 VSSFTWEESNAYYWQPYALPL 895 QPYALPL 896 Py-1-NapPYQJYALPL 897 TANVSSFEWTPG YWQPYALPL898 FEWTPGYWQPYALPL 899 FEWTPGYWQJYALPL 900 FEWTPGYYQJYALPL 901ETPFTWEESNAYYWQPYALPL 902 FTWEESNAYYWQJYALPL 903 ADVL YWQPYA PVTLWV 904GDVAE YWQPYA LPLTSL 905 SWTDYG YWQPYA LPISGL 906 FEWTPGYWQPYALPL 911FEWTPGYWQJYALPL 912 FEWTPGWYQPYALPL 913 FEWTPGWYQJYALPL 914FEWTPGYYQPYALPL 915 FEWTPGYYQJYALPL 916 TANVSSFEWTPGYWQPYALPL 918SWTDYGYWQPYALPISGL 919 ETPFTWEESNAYYWQPYALPL 920 ENTYSPNWADSMYWQPYALPL921 SVGEDHNFWTSEYWQPYALPL 922 DGYDRWRQSGERYWQPYALPL 923 FEWTPGYWQPYALPL924 FEWTPGYWQPY 925 FEWTPGYWQJY 926 EWTPGYWQPY 927 FEWTPGWYQJY 928AEWTPGYWQJY 929 FAWTPGYWQJY 930 FEATPGYWQJY 931 FEWAPGYWQJY 932FEWTAGYWQJY 933 FEWTPAYWQJY 934 FEWTPGAWQJY 935 FEWTPGYAQJY 936FEWTPGYWQJA 937 FEWTGGYWQJY 938 FEWTPGYWQJY 939 FEWTJGYWQJY 940FEWTPecGYWQJY 941 FEWTPAibYWQJY 942 FEWTPSarWYQJY 943 FEWTSarGYWQJY 944FEWTPNYWQJY 945 FEWTPVYWQJY 946 FEWTVPYWQJY 947 AcFEWTPGWYQJY 948AcFEWTPGYWQJY 949 INap-EWTPGYYQJY 950 YEWTPGYYQJY 951 FEWVPGYYQJY 952FEWTPGYYQJY 953 FEWTPsYYQJY 954 FEWTPnYYQJY 955 SHLY-Nap-QPYSVQM 956TLVY-Nap-QPYSLQT 957 RGDY-Nap-QPYSVQS 958 NMVY-Nap-QPYSIQT 959 VYWQPYSVQ960 VY-Nap-QPYSVQ 961 TFVYWQJYALPL 962 FEWTPGYYQJ-Bpa 963XaaFEWTPGYYQJ-Bpa 964 FEWTPGY-Bpa-QJY 965 AcFEWTPGY-Bpa-QJY 966FEWTPG-Bpa-YQJY 967 AcFEWTPG-Bpa-YQJY 968 AcFE-Bpa-TPGYYQJY 969AcFE-Bpa-TPGYYQJY 970 Bpa-EWTPGYYQJY 971 AcBpa-EWTPGYYQJY 972 VYWQPYSVQ973 RLVYWQPYSVQR 974 RLVY-Nap-QPYSVQR 975 RLDYWQPYSVQR 976 RLVWFQPYSVQR977 RLVYWQPYSIQR 978 DNSSWYDSFLL 980 DNTAWYESFLA 981 DNTAWYENFLL 982PARE DNTAWYDSFLI WC 983 TSEY DNTTWYEKFLA SQ 984 SQIP DNTAWYQSFLL HG 985SPFI DNTAWYENFLL TY 986 EQIY DNTAWYDHFLL SY 987 TPFI DNTAWYENFLL TY 988TYTY DNTAWYERFLM SY 989 TMTQ DNTAWYENFLL SY 990 TI DNTAWYANLVQ TYPQ 991TI DNTAWYERFLA QYPD 992 HI DNTAWYENFLL TYTP 993 SQ DNTAWYENFLL SYKA 994QI DNTAWYERFLL QYNA 995 NQ DNTAWYESFLL QYNT 996 TI DNTAWYENFLL NHNL 997HY DNTAWYERFLQ QGWH 998 ETPFTWEESNAYYWQPYALPL 999 YIPFTWEESNAYYWQPYALPL1000 DGYDRWRQSGERYWQPYALPL 1001 pY-INap-pY-QJYALPL 1002TANVSSFEWTPGYWQPYALPL 1003 FEWTPGYWQJYALPL 1004 FEWTPGYWQPYALPLSD 1005FEWTPGYYQJYALPL 1006 FEWTPGYWQJY 1007 AcFEWTPGYWQJY 1008 AcFEWTPGWYQJY1009 AcFEWTPGYYQJY 1010 AcFEWTPaYWQJY 1011 AcFEWTPaWYQJY 1012AcFEWTPaYYQJY 1013 FEWTPGYYQJYALPL 1014 FEWTPGYWQJYALPL 1015FEWTPGWYQJYALPL 1016 TANVSSFEWTPGYWQPYALPL 1017 AcFEWTPGYWQJY 1018AcFEWTPGWYQJY 1019 AcFEWTPGYYQJY 1020 AcFEWTPAYWQJY 1021 AcFEWTPAWYQJY1022 AcFEWTPAYYQJY 1023

TABLE 5 EPO-mimetic peptide sequences SEQ Sequence/structure ID NO:YXCXXGPXTWXCXP  83 YXCXXGPXTWXCXP-YXCXXGPXTWXCXP  84YXCXXGPXTWXCXP-Λ-YXCXXGPXTWXCXP  85

 86        86 GGTYSCHFGPLTWVCKPQGG  87 GGDYHCRMGPLTWVCKPLGG  88GGVYACRMGPITWVCSPLGG  89 VGNYMCHFGPITWVCRPGGG  90 GGLYLCRFGPVTWDCGYKGG 91 GGTYSCHFGPLTWVCKPQGG-  92 GGTYSCHFGPLTWVCKPQGGGGTYSCHFGPLTWVCKPQGG-Λ-  93 GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGGSSK 94 GGTYSCHFGPLTWVCKPQGGSSK-  95 GGTYSCHFGPLTWVCKPQGGSSKGGTYSCHFGPLTWVCKPQGGSSK-Λ-  96 GGTYSCHFGPLTWVCKPQGGSSK

 97       97 GGTYSCHFGPLTWVCKPQGGSSK(-Λ-biotin)  98 CX₄X₅GPX₆TWX₇C  421GGTYSCHGPLTWVCKPQGG  422 VGNYMAHMGPITWVCRPGG  423 GGPHHVYACRMGPLTWIC 424 GGTYSCHFGPLTWVCKPQ  425 GGLYACHMGPMTWVCQPLRG  426TIAQYICYMGPETWECRPSPKA  427 YSCHFGPLTWVCK  428 YCHFGPLTWVC  429X₃X₄X₅GPX₆TWX₇X₈  124 YX₂X₃X₄X₅GPX₆TWX₇X₈  461X₁YX₂X₃X₄X₅GPX₆TWX₇X₈X₉X₁₀X₁₁  419 X₁YX₂CX₄X₅GPX₆TWX₇CX₉X₁₀X₁₁  420GGLYLCRFGPVTWDCGYKGG 1024 GGTYSCHFGPLTWVCKPQGG 1025 GGDYHCRMGPLTWVCKPLGG1026 VGNYMCHFGPITWVCRPGGG 1029 GGVYACRMGPITWVCSPLGG 1030VGNYMAHMGPITWVCRPGG 1035 GGTYSCHFGPLTWVCKPQ 1036 GGLYACHMGPMTWVCQPLRG1037 TIAQYICYMGPETWECRPSPKA 1038 YSCHFGPLTWVCK 1039 YCHFGPLTWVC 1040SCHFGPLTWVCK 1041 (AX₂)_(n)X₃X₄X₅GPX₆TWX₇X₈ 1042

TABLE 6 TPO-mimetic peptide sequences SEQ Sequence/structure ID NO:IEGPTLRQWLAARA 13 IEGPTLRQWLAAKA 24 IEGPTLREWLAARA 25IEGPTLRQWLAARA-Λ-IEGPTLRQWLAARA 26 IEGPTLRQWLAAKA-Λ-IEGPTLRQWLAAKA 27

28 IEGPTLRQWLAARA-Λ-K(BrAc)-Λ-IEGPTLRQWLAARA 29IEGPTLRQWLAARA-Λ-K(PEG)-Λ-IEGPTLRQWLAARA 30

31 31

32 32 VRDQIXXXL 33 TLREWL 34 GRVRDQVAGW 35 GRVKDQIAQL 36 GVRDQVSWAL 37ESVREQVMKY 38 SVRSQISASL 39 GVRETVYRHM 40 GVREVIVMHML 41 GRVRDQIWAAL 42AGVRDQILIWL 43 GRVRDQIMLSL 44 GRVRDQI(X)₃L 45 CTLRQWLQGC 46 CTLQEFLEGC47 CTRTEWLHGC 48 CTLREWLHGGFC 49 CTLREWVFAGLC 50 CTLRQWLILLGMC 51CTLAEFLASGVEQC 52 CSLQEFLSHGGYVC 53 CTLREFLDPTTAVC 54 CTLKEWLVSHEVWC 55CTLREWL(X)_(2–6)C 56–60 REGPTLRQWM 61 EGPTLRQWLA 62 ERGPFWAKAC 63REGPRCVMWM 64 CGTEGPTLSTWLDC 65 CEQDGPTLLEWLKC 66 CELVGPSLMSWLTC 67CLTGPFVTQWLYEC 68 CRAGPTLLEWLTLC 69 CADGPTLREWISFC 70C(X)_(1–2)EGPTLREWL(X)_(1–2)C 71–74 GGCTLREWLHGGFCGG 75GGCADGPTLREWISFCGG 76 GNADGPTLRQWLEGRRPKN 77 LAIEGPTLRQWLHGNGRDT 78HGRVGPTLREWKTQVATKK 79 TIKGPTLRQWLKSREHTS 80 ISDGPTLKEWLSVTRGAS 81SIEGPTLREWLTSRTPHS 82

TABLE 7 G-CSF-mimetic peptide sequences SEQ Sequence/structure ID NO:EEDCK  99

 99  99 EEDσK 100

100 100 pGluEDσK 101

101 101 PicSDσK 102

102 102 EEDCK-Λ-EEDCK 103 EEDXK-Λ-EEDXK 104

TABLE 8 TNF-antagonist peptide sequences SEQ Sequence/structure ID NO:YCFTASENHCY 106 YCFTNSENHCY 107 YCFTRSENHCY 108 FCASENHCY 109 YCASENHCY110 FCNSENHCY 111 FCNSENRCY 112 FCNSVENRCY 113 YCSQSVSNDCF 114 FCVSNDRCY115 YCRKELGQVCY 116 YCKEPGQCY 117 YCRKEMGCY 118 FCRKEMGCY 119 YCWSQNLCY120 YCELSQYLCY 121 YCWSQNYCY 122 YCWSQYLCY 123 DFLPHYKNTSLGHRP 1085

NR

TABLE 9 Integrin-binding peptide sequences SEQ Sequence/structure ID NO:RX₁ETX₂WX₃ 441 RX₁ETX₂WX₃ 442 RGDGX 443 CRGDGXC 444 CX₁X₂RLDX₃X₄C 445CARRLDAPC 446 CPSRLDSPC 447 X₁X₂X₃RGDX₄X₅X₆ 448 CX₂CRGDCX₅C 449CDCRGDCFC 450 CDCRGDCLC 451 CLCRGDCIC 452 X₁X₂DDX₄X₅X₇X₈ 453X₁X₂X₃DDX₄X₅X₆X₇X₈ 454 CWDDGWLC 455 CWDDLWWLC 456 CWDDGLMC 457 CWDDGWMC458 CSWDDGWLC 459 CPDDLWWLC 460 NGR NR GSL NR RGD NR CGRECPRLCQSSC 1071CNGRCVSGCAGRC 1072 CLSGSLSC 1073 RGD NR NGR NR GSL NR NGRAHA 1074 CNGRC1075 CDCRGDCFC 1076 CGSLVRC 1077 DLXXL 1043 RTDLDSLRTYTL 1044 RTDLDSLRTY1053 RTDLDSLRT 1054 RTDLDSLR 1078 GDLDLLKLRLTL 1079 GDLHSLRQLLSR 1080RDDLHMLRLQLW 1081 SSDLHALKKRYG 1082 RGDLKQLSELTW 1083 RGDLAALSAPPV 1084

TABLE 10 Selectin antagonist peptide sequences SEQ Sequence/structure IDNO: DITWDQLWDLMK 147 DITWDELWKIMN 148 DYTWFELWDMMQ 149 QITWAQLWNMMK 150DMTWHDLWTLMS 151 DYSWHDLWEMMS 152 EITWDQLWEVMN 153 HVSWEQLWDIMN 154HITWDQLWRIMT 155 RNMSWLELWEHMK 156 AEWTWDQLWHVMNPAESQ 157 HRAEWLALWEQMSP158 KKEDWLALWRIMSV 159 ITWDQLWDLMK 160 DITWDQLWDLMK 161 DITWDQLWDLMK 162DITWDQLWDLMK 163 CQNRYTDLVAIQNKNE 462 AENWADNEPNNKRNNED 463RKNNKTWTWVGTKKALTNE 464 KKALTNEAENWAD 465 CQXRYTDLVAIQNKXE 466RKXNXXWTWVGTXKXLTEE 467 AENWADGEPNNKXNXED 468 CXXXYTXLVAIQNKXE 469RKXXXXWXWVGTXKXLTXE 470 AXNWXXXEPNNXXXED 471 XKXKTXEAXNWXX 472

TABLE 11 Antipathogenic peptide sequences SEQ Sequence/structure ID NO:GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ 503 GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE504 GFFALIPKIISSPLFKTLLSAV 505 GFFALIPKIISSPLFKTLLSAV 506KGFFALIPKIISSPLFKTLLSAV 507 KKGFFALIPKIISSPLFKTLLSAV 508KKGFFALIPKIISSPLFKTLLSAV 509 GFFALIPKIIS 510 GIGAVLKVLTTGLPALISWIKRKRQQ511 GIGAVLKVLTTGLPALISWIKRKRQQ 512 GIGAVLKVLTTGLPALISWIKRKRQQ 513GIGAVLKVLTTGLPALISWIKR 514 AVLKVLTTGLPALISWIKR 515 KLLLLLKLLLLK 516KLLLKLLLKLLK 517 KLLLKLKLKLLK 518 KKLLKLKLKLKK 519 KLLLKLLLKLLK 520KLLLKLKLKLLK 521 KLLLLK 522 KLLLKLLK 523 KLLLKLKLKLLK 524 KLLLKLKLKLLK525 KLLLKLKLKLLK 526 KAAAKAAAKAAK 527 KVVVKVVVKVVK 528 KVVVKVKVKVVK 529KVVVKVKVKVK 530 KVVVKVKVKVVK 531 KLILKL 532 KVLHLL 533 LKLRLL 534 KPLHLL535 KLILKLVR 536 KVFHLLHL 537 HKFRILKL 538 KPFHILHL 539 KIIIKIKIKIIK 540KIIIKIKIKIIK 541 KIIIKIKIKIIK 542 KIPIKIKIKIPK 543 KIPIKIKIKIVK 544RIIIRIRIRIIR 545 RIIIRIRIRIIR 546 RIIIRIRIRIIR 547 RIVIRIRIRLIR 548RIIVRIRLRIIR 549 RIGIRLRVRIIR 550 KIVIRIRIRLIR 551 RIAVKWRLRFIK 552KIGWKLRVRIIR 553 KKIGWLIIRVRR 554 RIVIRIRIRLIRIR 555 RIIVRIRLRIIRVR 556RIGIRLRVRIIRRV 557 KIVIRIRARLIRIRIR 558 RIIVKIRLRIIKKIRL 559KIGIKARVRIIRVKII 560 RIIVHIRLRIIHHIRL 561 HIGIKAHVRIIRVHII 562RIYVKIHLRYIKKIRL 563 KIGHKARVHIIRYKII 564 RIYVKPHPRYIKKIRL 565KPGHKARPHIIRYKII 566 KIVIRIRIRLIRIRIRKIV 567 RIIVKIRLRIIKKIRLIKK 568KIGWKLRVRIIRVKIGRLR 569 KIVIRIRIRLIRIRIRKIVKVKRIR 570RFAVKIRLRIIKKIRLIKKIRKRVIK 571 KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 572RIYVKPHPRYIKKIRL 573 KPGHKARPHIIRYKII 574 KIVIRIRIRLIRIRIRKIV 575RIIVKIRLRIIKKIRLIKK 576 RIYVSKISIYIKKIRL 577 KIVIFTRIRLTSIRIRSIV 578KPIHKARPTIIRYKMI 579 cyclicCKGFFALIPKIISSPLFKTLLSAVC 580CKKGFFALIPKIISSPLFKTLLSAVC 581 CKKKGFFALIPKIISSPLFKTLLSAVC 582CyclicCRIVIRIRIRLIRIRC 583 CyclicCKPGHKARPHIIRYKIIC 584CyclicCRFAVKIRLRIIKKIRLIKKIRKRVIKC 585 KLLLKLLL KLLKC 586 KLLLKLLLKLLK587 KLLLKLKLKLLKC 588 KLLLKLLLKLLK 589

TABLE 12 VIP-mimetic peptide sequences SEQ Sequence/structure ID NO:HSDAVFYDNYTR LRKQMAVKKYLN SILN 590 Nle HSDAVFYDNYTR LRKQMAVKKYLN SILN591 X₁ X₁′ X₁″ X₂ 592 X₃ S X₄ LN 593

594 KKYL 595 NSILN 596 KKYL 597 KKYA 598 AVKKYL 599 NSILN 600 KKYV 601SILauN 602 KKYLNle 603 NSYLN 604 NSIYN 605 KKYLPPNSILN 606 LauKKYL 607CapKKYL 608 KYL NR KKYNle 609 VKKYL 610 LNSILN 611 YLNSILN 612 KKYLN 613KKYLNS 614 KKYLNSI 615 KKYLNSIL 616 KKYL 617 KKYDA 618 AVKKYL 619 NSILN620 KKYV 621 SILauN 622 NSYLN 623 NSIYN 624 KKYLNle 625 KKYLPPNSILN 626KKYL 627 KKYDA 628 AVKKYL 629 NSILN 630 KKYV 631 SILauN 632 LauKKYL 633CapKKYL 634 KYL NR KYL NR KKYNle 635 VKKYL 636 LNSILN 637 YLNSILN 638KKYLNIe 639 KKYLN 640 KKYLNS 641 KKYLNSI 642 KKYLNSIL 643 KKKYLD 644cyclicCKKYLC 645

646 KKYA 647 WWTDTGLW 648 WWTDDGLW 649 WWDTRGLWVWTI 650 FWGNDGIWLESG 651DWDQFGLWRGAA 652 RWDDNGLWVVVL 653 SGMWSHYGIWMG 654 GGRWDQAGLWVA 655KLWSEQGIWMGE 656 CWSMHGLWLC 657 GCWDNTGIWVPC 658 DWDTRGLWVY 659SLWDENGAWI 660 KWDDRGLWMH 661 QAWNERGLWT 662 QWDTRGLWVA 663 WNVHGIWQE664 SWDTRGLWVE 665 DWDTRGLWVA 666 SWGRDGLWIE 667 EWTDNGLWAL 668SWDEKGLWSA 669 SWDSSGLWMD 670

TABLE 13 Mdm/hdm antagonist peptide sequences SEQ Sequence/structure IDNO: TFSDLW 130 QETFSDLWKLLP 131 QPTFSDLWKLLP 132 QETFSDYWKLLP 133QPTFSDYWKLLP 134 MPRFMDYWEGLN 135 VQNFIDYWTQQF 136 TGPAFTHYWATF 137IDRAPTFRDHWFALV 138 PRPALVFADYWETLY 139 PAFSRFWSDLSAGAH 140PAFSRFWSKLSAGAH 141 PXFXDYWXXL 142 QETFSDLWKLLP 143 QPTFSDLWKLLP 144QETFSDYWKLLP 145 QPTFSDYWKLLP 146

TABLE 14 Calmodulin antagonist peptide sequences SEQ Sequence/structureID NO: SCVKWGKKEFCGS 164 SCWKYWGKECGS 165 SCYEWGKLRWCGS 166SCLRWGKWSNCGS 167 SCWRWGKYQICGS 168 SCVSWGALKLCGS 169 SCIRWGQNTFCGS 170SCWQWGNLKICGS 171 SCVRWGQLSICGS 172 LKKFNARRKLKGAILTTMLAK 173RRWKKNFIAVSAANRFKK 174 RKWQKTGHAVRAIGRLSS 175 INLKALAALAKKIL 176KIWSILAPLGTTLVKLVA 177 LKKLLKLLKKLLKL 178 LKWKKLLKLLKKLLKKLL 179AEWPSLTEIKTLSHFSV 180 AEWPSPTRVISTTYFGS 181 AELAHWPPVKTVLRSFT 182AEGSWLQLLNLMKQMNN 183 AEWPSLTEIK 184

TABLE 15 Mast cell antagonists/Mast cell protease inhibitor peptidesequences SEQ Sequence/structure ID NO: SGSGVLKRPLPILPVTR 272RWLSSRPLPPLPLPPRT 273 GSGSYDTLALPSLPLHPMSS 274 GSGSYDTRALPSLPLHPMSS 275GSGSSGVTMYPKLPPHWSMA 276 GSGSSGVRMYPKLPPHWSMA 277 GSGSSSMRMVPTIPGSAKHG278 RNR NR QT NR RQK NR NRQ NR RQK NR RNRQKT 436 RNRQ 437 RNRQK 438NRQKT 439 RQKT 440

TABLE 16 SH3 antagonist peptide sequences SEQ Sequence/structure ID NO:RPLPPLP 282 RELPPLP 283 SPLPPLP 284 GPLPPLP 285 RPLPIPP 286 RPLPIPP 287RRLPPTP 288 RQLPPTP 289 RPLPSRP 290 RPLPTRP 291 SRLPPLP 292 RALPSPP 293RRLPRTP 294 RPVPPIT 295 ILAPPVP 296 RPLPMLP 297 RPLPILP 298 RPLPSLP 299RPLPSLP 300 RPLPMIP 301 RPLPLIP 302 RPLPPTP 303 RSLPPLP 304 RPQPPPP 305RQLPIPP 306 XXXRPLPPLPXP 307 XXXRPLPPIPXX 308 XXXRPLPPLPXX 309RXXRPLPPLPXP 310 RXXRPLPPLPPP 311 PPPYPPPPIPXX 312 PPPYPPPPVPXX 313LXXRPLPXΨP 314 ΨXXRPLPXLP 315 PPXΘXPPPΨP 316 +PPΨPPXKPXWL 317 RPXΨPΨR+ SXP 318 PPVPPRPXXTL 319 ΨPΨLPΨK 320 +ΘDXPLPXLP 321

TABLE 17 Somatostatin or cortistatin mimetic peptide sequences SEQSequence/structure ID NO: X¹-X²-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X³-Ser-X⁴473 Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys474 Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 475 CysArg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 476 Asp Arg Met Pro CysArg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 477 Met Pro Cys Arg Asn PhePhe Trp Lys Thr Phe Ser Ser Cys 478 Cys Arg Asn Phe Phe Trp Lys Thr PheSer Ser Cys 479 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe SerSer Cys 480 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys481 Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 482 Asp Arg MetPro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 483 Met Pro Cys LysAsn Phe Phe Trp Lys Thr Phe Ser Ser Cys 484 Cys Lys Asn Phe Phe Trp LysThr Phe Ser Ser Cys 485 Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys ThrPhe Thr Ser Cys Lys 486 Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe ThrSer Cys Lys 487 Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 488Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 489 MetPro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 490 Cys Arg Asn PhePhe Trp Lys Thr Phe Thr Ser Cys 491 Asp Arg Met Pro Cys Lys Asn Phe PheTrp Lys Thr Phe Thr Ser Cys Lys 492 Met Pro Cys Lys Asn Phe Phe Trp LysThr Phe Thr Ser Cys Lys 493 Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr SerCys Lys 494 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr SerCys 495 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 496 CysLys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 497

TABLE 18 UKR antagonist peptide sequences SEQ Sequence/structure ID NO:AEPMPHSLNFSQYLWYT 196 AEHTYSSLWDTYSPLAF 197 AELDLWMRHYPLSFSNR 198AESSLWTRYAWPSMPSY 199 AEWHPGLSFGSYLWSKT 200 AEPALLNWSFFFNPGLH 201AEWSFYNLHLPEPQTIF 202 AEPLDLWSLYSLPPLAM 203 AEPTLWQLYQFPLRLSG 204AEISFSELMWLRSTPAF 205 AELSEADLWTTWFGMGS 206 AESSLWRIFSPSALMMS 207AESLPTLTSILWGKESV 208 AETLFMDLWHDKHILLT 209 AEILNFPLWHEPLWSTE 210AESQTGTLNTLFWNTLR 211 AEPVYQYELDSYLRSYY 430 AELDLSTFYDIQYLLRT 431AEFFKLGPNGYVYLHSA 432 FKLXXXGYVYL 433 AESTYHHLSLGYMYTLN 434 YHXLXXGYMYT435

TABLE 19 Macrophage and/or T-cell inhibiting peptide sequences SEQSequence/structure ID NO: Xaa-Yaa-Arg NR Arg-Yaa-Xaa NR Xaa-Arg-Yaa NRYaa-Arg-Xaa NR Ala-Arg NR Arg-Arg NR Asn-Arg NR Asp-Arg NR Cys-Arg NRGln-Arg NR Glu-Arg NR Gly-Arg NR His-arg NR Ile-Arg NR Leu-Arg NRLys-Arg NR Met-Arg NR Phe-Arg NR Ser-Arg NR Thr-Arg NR Trp-Arg NRTyr-Arg NR Val-Arg NR Ala-Glu-Arg NR Arg-Glu-Arg NR Asn-Glu-Arg NRAsp-Glu-Arg NR Cys-Glu-Arg NR Gln-Glu-Arg NR Glu-Glu-Arg NR Gly-Glu-ArgNR His-Glu-Arg NR Ile-Glu-Arg NR Leu-Glu-Arg NR Lys-Glu-Arg NRMet-Glu-Arg NR Phe-Glu-Arg NR Pro-Glu-Arg NR Ser-Glu-Arg NR Thr-Glu-ArgNR Trp-Glu-Arg NR Tyr-Glu-Arg NR Val-Glu-Arg NR Arg-Ala NR Arg-Asp NRArg-Cys NR Arg-Gln NR Arg-Glu NR Arg-Gly NR Arg-His NR Arg-Ile NRArg-Leu NR Arg-Lys NR Arg-Met NR Arg-Phe NR Arg-Pro NR Arg-Ser NRArg-Thr NR Arg-Trp NR Arg-Tyr NR Arg-Val NR Arg-Glu-Ala NR Arg-Glu-AsnNR Arg-Glu-Asp NR Arg-Glu-Cys NR Arg-Glu-Gln NR Arg-Glu-Glu NRArg-Glu-Gly NR Arg-Glu-His NR Arg-Glu-Ile NR Arg-Glu-Leu NR Arg-Glu-LysNR Arg-Glu-Met NR Arg-Glu-Phe NR Arg-Glu-Pro NR Arg-Glu-Ser NRArg-Glu-Thr NR Arg-Glu-Trp NR Arg-Glu-Tyr NR Arg-Glu-Val NR Ala-Arg-GluNR Arg-Arg-Glu NR Asn-Arg-Glu NR Asp-Arg-Glu NR Cys-Arg-Glu NRGln-Arg-Glu NR Glu-Arg-Glu NR Gly-Arg-Glu NR His-Arg-Glu NR Ile-Arg-GluNR Leu-Arg-Glu NR Lys-Arg-Glu NR Met-Arg-Glu NR Phe-Arg-Glu NRPro-Arg-Glu NR Ser-Arg-Glu NR Thr-Arg-Glu NR Trp-Arg-Glu NR Tyr-Arg-GluNR Val-Arg-Glu NR Glu-Arg-Ala, NR Glu-Arg-Arg NR Glu-Arg-Asn NRGlu-Arg-Asp NR Glu-Arg-Cys NR Glu-Arg-Gln NR Glu-Arg-Gly NR Glu-Arg-HisNR Glu-Arg-Ile NR Glu-Arg-Leu NR Glu-Arg-Lys NR Glu-Arg-Met NRGlu-Arg-Phe NR Glu-Arg-Pro NR Glu-Arg-Ser NR Glu-Arg-Thr NR Glu-Arg-TrpNR Glu-Arg-Tyr NR Glu-Arg-Val NR

TABLE 20 Additional Fxemplary Pharmacologically Active Peptides SEQ IDSequence/structure NO: Activity VEPNCDIHVMWEWECFERL 1027 VEGF-antagonistGERWCFDGPLTWVCGEES 1084 VEGF-antagonist RGWVEICVADDNGMCVTEAQ 1085VEGF-antagonist GWDECDVARMWEWECFAGV 1086 VEGF-antagonistGERWCFDGPRAWVCGWEI 501 VEGF-antagonist EELWCFDGPRAWVCGYVK 502VEGF-antagonist RGWVEICAADDYGRCLTEAQ 1031 VEGF-antagonistRGWVEICESDVWGRCL 1087 VEGF-antagonist RGWVEICESDVWGRCL 1088VEGF-antagonist GGNECDIARMWEWECFERL 1089 VEGF-antagonistRGWVEICAADDYGRCL 1090 VEGF-antagonist CTTHWGFTLC 1028 MMP inhibitorCLRSGXGC 1091 MMP inhibitor CXXHWGFXXC 1092 MMP inhibitor CXPXC 1093 MMPinhibitor CRRHWGFEFC 1094 MMP inhibitor STTHWGFTLS 1095 MMP inhibitorCSLHWGFWWC 1096 CTLA4-mimetic GFVCSGIFAVGVGRC 125 CTLA4-mimeticAPGVRLGCAVLGRYC 126 CTLA4-mimetic LLGRMK 105 Antiviral (HBV)ICVVQDWGHHRCTAGHMANLTSHASAI 127 C3b antagonist ICVVQDWGHHRCT 128 C3bantagonist CVVQDWGHHAC 129 C3b antagonist STGGFDDVYDWARGVSSALTTTLVATR185 Vinculin-binding STGGFDDVYDWARRVSSALTTTLVATR 186 Vinculin-bindingSRGVNFSEWLYDMSAAMKEASNVFPSRRSR 187 Vinculin-bindingSSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 188 Vinculin-bindingSSPSLYTQFLVNYESAATRIQDLLIASRPSR 189 Vinculin-bindingSSTGWVDLLGALQRAADATRTSIPPSLQNSR 190 Vinculin-binding DVYTKKELIECARRVSEK191 Vinculin-binding EKGSYYPGSGIAQFHIDYNNVS 192 C4BP-bindingSGIAQFHIDYNNVSSAEGWHVN 193 C4BP-bindingLVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN 194 C4BP-binding SGIAQFHIDYNNVS 195C4BP-binding LLGRMK 279 anti-HBV ALLGRMKG 280 anti-HBV LDPAFR 281anti-HBV CXXRGDC 322 Inhibition of platelet aggregation RPLPPLP 323 Srcantagonist PPVPPR 324 Src antagonist XFXDXWXXLXX 325 Anti-cancer(particularly for sarcomas) KACRRLFGPVDSEQLSRDCD 326 p16-mimeticRERWNFDFVTETPLEGDFAW 327 p16-mimetic KRRQTSMTDFYHSKRRLIFS 328p16-mimetic TSMTDFYHSKRRLIFSKRKP 329 p16-mimetic RRLIF 330 p16-mimeticKRRQTSATDFYHSKRRLIFSRQIKIWFQNRRMKWKK 331 p16-mimeticKRRLIFSKRQIKIWFQNRRMKWKK 332 p16-mimetic Asn Gln Gly Arg His Phe Cys GlyGly Ala Leu Ile His Ala 498 CAP37 mimetic/LPS Arg Phe Val Met Thr AlaAla Ser Cys Phe Gln binding Arg His Phe Cys Gly Gly Ala Leu Ile His AlaArg Phe Val 499 CAP37 mimetic/LPS Met Thr Ala Ala Ser Cys binding GlyThr Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg Ser 500 CAP37mimetic/LPS Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val Asn Val bindingWHWRHRIPLQLAAGR 1097 carbohydrate (GD1 alpha) mimetic LKTPRV 1098 β2GPIAb binding NTLKTPRV 1099 β2GPI Ab binding NTLKTPRVGGC 1100 β2GPI Abbinding KDKATF 1101 β2GPI Ab binding KDKATFGCHD 1102 β2GPI Ab bindingKDKATFGCHDGC 1103 β2GPI Ab binding TLRVYK 1104 β2GPI Ab bindingATLRVYKGG 1105 β2GPI Ab binding CATLRVYKGG 1106 β2GPI Ab bindingINLKALAALAKKIL 1107 Membrane- transporting GWT NR Membrane- transportingGWTLNSAGYLLG 1108 Membrane- transporting GWTLNSAGYLLGKINLKALAALAKKIL1109 Membrane- transporting

The present invention is also particularly useful with peptides havingactivity in treatment of:

-   cancer, wherein the peptide is a VEGF-mimetic or a VEGF receptor    antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the    like;-   asthma, wherein the protein of interest is a CKR3 antagonist, an    IL-5 receptor antagonist and the like;-   thrombosis, wherein the protein of interest is a GpIIb antagonist, a    GPIIIa antagonist, and the like;-   autoimmune diseases and other conditions involving immune    modulation, wherein the protein of interest is an IL-2 receptor    antagonist, a CD40 agonist or antagonist, a CD40L agonist or    antagonist, a thymopoietin mimetic and the like.

Vehicles. This invention requires the presence of at least one vehicle(F¹, F²) attached to a peptide through the N-terminus, C-terminus or asidechain of one of the amino acid residues. Multiple vehicles may alsobe used; e.g., Fc's at each terminus or an Fc at a terminus and a PEGgroup at the other terminus or a sidechain.

An Fc domain is the preferred vehicle. The Fc domain may be fused to theN or C termini of the peptides or at both the N and C termini. For theTPO-mimetic peptides, molecules having the Fc domain fused to the Nterminus of the peptide portion of the molecule are more bioactive thanother such fusions, so fusion to the N terminus is preferred.

As noted above, Fc variants are suitable vehicles within the scope ofthis invention. A native Fc may be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

-   1. Sites involved in disulfide bond formation are removed. Such    removal may avoid reaction with other cysteine-containing proteins    present in the host cell used to produce the molecules of the    invention. For this purpose, the cysteine-containing segment at the    N-terminus may be truncated or cysteine residues may be deleted or    substituted with other amino acids (e.g., alanyl, seryl). In    particular, one may truncate the N-terminal 20-amino acid segment of    SEQ ID NO: 2 or delete or substitute the cysteine residues at    positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are    removed, the single chain Fc domains can still form a dimeric Fc    domain that is held together non-covalently.-   2. A native Fc is modified to make it more compatible with a    selected host cell. For example, one may remove the PA sequence near    the N-terminus of a typical native Fc, which may be recognized by a    digestive enzyme in E. coli such as proline iminopeptidase. One may    also add an N-terminal methionine residue, especially when the    molecule is expressed recombinantly in a bacterial cell such as E.    coli. The Fc domain of SEQ ID NO: 2 (FIG. 4) is one such Fc variant.-   3. A portion of the N-terminus of a native Fc is removed to prevent    N-terminal heterogeneity when expressed in a selected host cell. For    this purpose, one may delete any of the first 20 amino acid residues    at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.-   4. One or more glycosylation sites are removed. Residues that are    typically glycosylated (e.g., asparagine) may confer cytolytic    response. Such residues may be deleted or substituted with    unglycosylated residues (e.g., alanine).-   5. Sites involved in interaction with complement, such as the C1q    binding site, are removed. For example, one may delete or substitute    the EKK sequence of human IgG1. Complement recruitment may not be    advantageous for the molecules of this invention and so may be    avoided with such an Fc variant.-   6. Sites are removed that affect binding to Fc receptors other than    a salvage receptor. A native Fc may have sites for interaction with    certain white blood cells that are not required for the fusion    molecules of the present invention and so may be removed.-   7. The ADCC site is removed. ADCC sites are known in the art; see,    for example, Molec. Immunol. 29 (5): 633–9 (1992) with regard to    ADCC sites in IgG1. These sites, as well, are not required for the    fusion molecules of the present invention and so may be removed.-   8. When the native Fc is derived from a non-human antibody, the    native Fc may be humanized. Typically, to humanize a native Fc, one    will substitute selected residues in the non-human native Fc with    residues that are normally found in human native Fc. Techniques for    antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2 (FIG. 4)the leucine at position 15 may be substituted with glutamate; theglutamate at position 99, with alanine; and the lysines at positions 101and 103, with alanines. In addition, one or more tyrosine residues canbe replaced by phenyalanine residues.

An alternative vehicle would be a protein, polypeptide, peptide,antibody, antibody fragment, or small molecule (e.g., a peptidomimeticcompound) capable of binding to a salvage receptor. For example, onecould use as a vehicle a polypeptide as described in U.S. Pat. No.5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also beselected by phage display for binding to the FcRn salvage receptor. Suchsalvage receptor-binding compounds are also included within the meaningof “vehicle” and are within the scope of this invention. Such vehiclesshould be selected for increased half-life (e.g., by avoiding sequencesrecognized by proteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

As noted above, polymer vehicles may also be used for F¹ and F². Variousmeans for attaching chemical moieties useful as vehicles are currentlyavailable, see, e.g., Patent Cooperation Treaty (“PCT”) InternationalPublication No. WO 96/11953, entitled “N-Terminally Chemically ModifiedProtein Compositions and Methods,” herein incorporated by reference inits entirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be linear or branched.The average molecular weight of the PEG will preferably range from about2 kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDato about 50 kDa, most preferably from about 5 kDa to about 10 kDa. ThePEG groups will generally be attached to the compounds of the inventionvia acylation or reductive alkylation through a reactive group on thePEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the inventive compound (e.g., an aldehyde, amino, orester group).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis (see, for example, FIGS. 5 and 6 andthe accompanying text herein). The peptides are “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1–6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference. Dextran of about 1 kD to about 20 kD is preferred whendextran is used as a vehicle in accordance with the present invention.

Linkers. Any “linker” group is optional. When present, its chemicalstructure is not critical, since it serves primarily as a spacer. Thelinker is preferably made up of amino acids linked together by peptidebonds. Thus, in preferred embodiments, the linker is made up of from 1to 20 amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅),poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:

(Gly)₃Lys(Gly)₄; (SEQ ID NO:333) (Gly)₃AsnGlySer(Gly)₂; (SEQ ID NO:334)(Gly)₃Cys(Gly)₄; and (SEQ ID NO:335) GlyProAsnGlyGly. (SEQ ID NO:336)To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are alsopreferred. The linkers shown here are exemplary; linkers within thescope of this invention may be much longer and may include otherresidues.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2–20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁–C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

Derivatives. The inventors also contemplate derivatizing the peptideand/or vehicle portion of the compounds. Such derivatives may improvethe solubility, absorption, biological half life, and the like of thecompounds. The moieties may alternatively eliminate or attenuate anyundesirable side-effect of the compounds and the like. Exemplaryderivatives include compounds in which:

-   1. The compound or some portion thereof is cyclic. For example, the    peptide portion may be modified to contain two or more Cys residues    (e.g., in the linker), which could cyclize by disulfide bond    formation. For citations to references on preparation of cyclized    derivatives, see Table 2.-   2. The compound is cross-linked or is rendered capable of    cross-linking between molecules. For example, the peptide portion    may be modified to contain one Cys residue and thereby be able to    form an intermolecular disulfide bond with a like molecule. The    compound may also be cross-linked through its C-terminus, as in the    molecule shown below.

-   4. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a    non-peptidyl linkage. Exemplary non-peptidyl linkages are    —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide    [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and    alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].-   5. The N-terminus is derivatized. Typically, the N-terminus may be    acylated or modified to a substituted amine. Exemplary N-terminal    derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹,    —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or    benzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R¹ are each    independently hydrogen or lower alkyl and wherein the phenyl ring    may be substituted with 1 to 3 substituents selected from the group    consisting of C₁–C₄ alkyl, C₁–C₄ alkoxy, chloro, and bromo.-   6. The free C-terminus is derivatized. Typically, the C-terminus is    esterified or amidated. For example, one may use methods described    in the art to add (NH—CH₂—CH₂—NH₂)₂ to compounds of this invention    having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise,    one may use methods described in the art to add —NH₂ to compounds of    this invention having any of SEQ ID NOS: 924 to 955, 963 to 972,    1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary    C-terminal derivative groups include, for example, —C(O)R² wherein    R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently    hydrogen or C₁–C₈ alkyl (preferably C₁–C₄ alkyl).-   7. A disulfide bond is replaced with another, preferably more    stable, cross-linking moiety (e.g., an alkylene). See, e.g.,    Bhatnagar et al. (1996), J. Med. Chem. 39: 3814–9; Alberts et    al. (1993) Thirteenth Am. Pep. Symp., 357–9.-   8. One or more individual amino acid residues is modified. Various    derivatizing agents are known to react specifically with selected    sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814–9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79–86 (1983).

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Methods of Making

The compounds of this invention largely may be made in transformed hostcells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of effecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal binding sites, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335–61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394–414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105–253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257–527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides.

Compounds that contain derivatized peptides or which contain non-peptidegroups may be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In general. The compounds of this invention have pharmacologic activityresulting from their ability to bind to proteins of interest asagonists, mimetics or antagonists of the native ligands of such proteinsof interest. The utility of specific compounds is shown in Table 2. Theactivity of these compounds can be measured by assays known in the art.For the TPO-mimetic and EPO-mimetic compounds, in vivo assays arefurther described in the Examples section herein.

In addition to therapeutic uses, the compounds of the present inventionare useful in diagnosing diseases characterized by dysfunction of theirassociated protein of interest. In one embodiment, a method of detectingin a biological sample a protein of interest (e.g., a receptor) that iscapable of being activated comprising the steps of: (a) contacting thesample with a compound of this invention; and (b) detecting activationof the protein of interest by the compound. The biological samplesinclude tissue specimens, intact cells, or extracts thereof. Thecompounds of this invention may be used as part of a diagnostic kit todetect the presence of their associated proteins of interest in abiological sample. Such kits employ the compounds of the inventionhaving an attached label to allow for detection. The compounds areuseful for identifying normal or abnormal proteins of interest. For theEPO-mimetic compounds, for example, presence of abnormal protein ofinterest in a biological sample may be indicative of such disorders asDiamond Blackfan anemia, where it is believed that the EPO receptor isdysfunctional.

Therapeutic uses of EPO-mimetic compounds. The EPO-mimetic compounds ofthe invention are useful for treating disorders characterized by low redblood cell levels. Included in the invention are methods of modulatingthe endogenous activity of an EPO receptor in a mammal, preferablymethods of increasing the activity of an EPO receptor. In general, anycondition treatable by erythropoietin, such as anemia, may also betreated by the EPO-mimetic compounds of the invention. These compoundsare administered by an amount and route of delivery that is appropriatefor the nature and severity of the condition being treated and may beascertained by one skilled in the art. Preferably, administration is byinjection, either subcutaneous, intramuscular, or intravenous.

Therapeutic uses of TPO-mimetic compounds. For the TPO-mimeticcompounds, one can utilize such standard assays as those described inWO95/26746 entitled “Compositions and Methods for StimulatingMegakaryocyte Growth and Differentiation”. In vivo assays also appear inthe Examples hereinafter.

The conditions to be treated are generally those that involve anexisting megakaryocyte/platelet deficiency or an expectedmegakaryocyte/platelet deficiency (e.g., because of planned surgery orplatelet donation). Such conditions will usually be the result of adeficiency (temporary or permanent) of active Mpl ligand in vivo. Thegeneric term for platelet deficiency is thrombocytopenia, and hence themethods and compositions of the present invention are generallyavailable for treating thrombocytopenia in patients in need thereof.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy and other therapy with a variety ofdrugs, radiation therapy, surgery, accidental blood loss, and otherspecific disease conditions. Exemplary specific disease conditions thatinvolve thrombocytopenia and may be treated in accordance with thisinvention are: aplastic anemia, idiopathic thrombocytopenia, metastatictumors which result in thrombocytopenia, systemic lupus erythematosus,splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic aciddeficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, andparoxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDSresult in thrombocytopenia (e.g., AZT). Certain wound healing disordersmight also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery, a compound of the present invention could be administeredseveral days to several hours prior to the need for platelets. Withregard to acute situations, e.g., accidental and massive blood loss, acompound of this invention could be administered along with blood orpurified platelets.

The TPO-mimetic compounds of this invention may also be useful instimulating certain cell types other than megakaryocytes if such cellsare found to express Mpl receptor. Conditions associated with such cellsthat express the Mpl receptor, which are responsive to stimulation bythe Mpl ligand, are also within the scope of this invention.

The TPO-mimetic compounds of this invention may be used in any situationin which production of platelets or platelet precursor cells is desired,or in which stimulation of the c-Mpl receptor is desired. Thus, forexample, the compounds of this invention may be used to treat anycondition in a mammal wherein there is a need of platelets,megakaryocytes, and the like. Such conditions are described in detail inthe following exemplary sources: WO95/26746; WO95/21919; WO95/18858;WO95/21920 and are incorporated herein.

The TPO-mimetic compounds of this invention may also be useful inmaintaining the viability or storage life of platelets and/ormegakaryocytes and related cells. Accordingly, it could be useful toinclude an effective amount of one or more such compounds in acomposition containing such cells.

The therapeutic methods, compositions and compounds of the presentinvention may also be employed, alone or in combination with othercytokines, soluble Mpl receptor, hematopoietic factors, interleukins,growth factors or antibodies in the treatment of disease statescharacterized by other symptoms as well as platelet deficiencies. It isanticipated that the inventive compound will prove useful in treatingsome forms of thrombocytopenia in combination with general stimulatorsof hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocyticstimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemiainhibitory factor (LIF), oncostatin M (OSM), or other molecules withmegakaryocyte stimulating activity may also be employed with Mpl ligand.Additional exemplary cytokines or hematopoietic factors for suchco-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5,IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It mayfurther be useful to administer, either simultaneously or sequentially,an effective amount of a soluble mammalian Mpl receptor, which appearsto have an effect of causing megakaryocytes to fragment into plateletsonce the megakaryocytes have reached mature form. Thus, administrationof an inventive compound (to enhance the number of maturemegakaryocytes) followed by administration of the soluble Mpl receptor(to inactivate the ligand and allow the mature megakaryocytes to produceplatelets) is expected to be a particularly effective means ofstimulating platelet production. The dosage recited above would beadjusted to compensate for such additional components in the therapeuticcomposition. Progress of the treated patient can be monitored byconventional methods.

In cases where the inventive compounds are added to compositions ofplatelets and/or megakaryocytes and related cells, the amount to beincluded will generally be ascertained experimentally by techniques andassays known in the art. An exemplary range of amounts is 0.1 μg–1 mginventive compound per 10⁶ cells.

Pharmaceutical Compositions

In General. The present invention also provides methods of usingpharmaceutical compositions of the inventive compounds. Suchpharmaceutical compositions may be for administration for injection, orfor oral, pulmonary, nasal, transdermal or other forms ofadministration. In general, the invention encompasses pharmaceuticalcompositions comprising effective amounts of a compound of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hyaluronic acid may also be used, and this may havethe effect of promoting sustained duration in the circulation. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435–1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder, such as lyophilized form.Implantable sustained release formulations are also contemplated, as aretransdermal formulations.

Oral dosage forms. Contemplated for use herein are oral solid dosageforms, which are described generally in Chapter 89 of Remington'sPharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa.18042, which is herein incorporated by reference. Solid dosage formsinclude tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Chapter 10of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker andC. T. Rhodes, herein incorporated by reference. In general, theformulation will include the inventive compound, and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the aboveinventive compounds. If necessary, the compounds may be chemicallymodified so that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe compound molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached vehicles in this invention may also be used forthis purpose. Examples of such moieties include: PEG, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, forexample, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymesas Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, NewYork, N.Y., pp 367–83; Newmark, et al. (1982), J. Appl. Biochem.4:185–9. Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See US Pat. No.5,792,451, “Oral drug delivery composition and methods”.

The compounds of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake ofthe compound. Additives potentially having this property are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of thisinvention could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compounds of this invention is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary delivery forms. Also contemplated herein is pulmonary deliveryof the present protein (or derivatives thereof). The protein (orderivative) is delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. (Otherreports of this include Adjei et al., Pharma. Res. (1990) 7: 565–9;Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135–44 (leuprolideacetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5):s.143–146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3:206–12 ((α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84:1145–6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482–8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. Nasal delivery of the inventive compound is alsocontemplated. Nasal delivery allows the passage of the protein to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran. Delivery via transport across other mucous membranes isalso contemplated.

Dosages. The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g. the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. Generally, the daily regimen should be in the range of 0.1–1000micrograms of the inventive compound per kilogram of body weight,preferably 0.1–150 micrograms per kilogram.

Specific Preferred Embodiments

The inventors have determined preferred peptide sequences for moleculeshaving many different kinds of activity. The inventors have furtherdetermined preferred structures of these preferred peptides combinedwith preferred linkers and vehicles. Preferred structures for thesepreferred peptides listed in Table 21 below.

TABLE 21 Preferred embodiments SEQ ID Sequence/structure NO: ActivityF¹-(G)₅-IEGPTLRQWLAARA-(G)₈-IEGPTLRQWLAARA 337 TPO-mimeticIEGPTLRQWLAARA-(G)₈-IEGPTLRQWLAARA-(G)₅-F¹ 338 TPO-mimeticF¹-(G)₅-IEGPTLRQWLAARA 1032 TPO-mimetic IEGPTLRQWLAARA-(G)₅-F¹ 1033TPO-mimetic F¹-(G)₅-GGTYSCHFGPLTWVCKPQGG-(G)₄- 339 EPO-mimeticGGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGG-(G)₄- 340 EPO-mimeticGGTYSCHFGPLTWVCKPQGG-(G)₅-F¹ GGTYSGHFGPLTWVCKPQGG-(G)₅-F¹ 1034EPO-mimetic F¹-(G)₅-DFLPHYKNTSLGHRP 1045 TNF-α inhibitorDFLPHYKNTSLGHRP-(G)₅-F¹ 1046 TNF-α inhibitor F¹-(G)₅-FEWTPGYWQPYALPL1047 IL-1 R antagonist FEWTPGYWQPYALPL-(G)₅-F¹ 1048 IL-1 R antagonistF¹-(G)₅-VEPNCDIHVMWEWECFERL 1049 VEGF-antagonistVEPNCDIHVMWEWECFERL-(G)₅-F¹ 1050 VEGF-antagonist F¹-(G)₅-CTTHWGFTLC 1051MMP inhibitor CTTHWGFTLC-(G)₅-F¹ 1052 MMP inhibitor “F¹” is an Fc domainas defined previously herein.

WORKING EXAMPLES

The compounds described above may be prepared as described below. Theseexamples comprise preferred embodiments of the invention and areillustrative rather than limiting.

Example 1 TPO-Mimetics

The following example uses peptides identified by the numbers appearingin Table A hereinafter.

Preparation of pep tide 19. Peptide 17b (12 mg) and MeO-PEG-SH 5000 (30mg, 2 equiv.) were dissolved in 1 ml aqueous buffer (pH 8). The mixturewas incubated at RT for about 30 minutes and the reaction was checked byanalytical HPLC, which showed a >80% completion of the reaction. Thepegylated material was isolated by preparative HPLC.

Preparation of peptide 20. Peptide 18 (14 mg) and MeO-PEG-maleimide (25mg) were dissolved in about 1.5 ml aqueous buffer (pH 8). The mixturewas incubated at RT for about 30 minutes, at which time about 70%transformation was complete as monitored with analytical HPLC byapplying an aliquot of sample to the HPLC column. The pegylated materialwas purified by preparative HPLC.

Bioactivity assay. The TPO in vitro bioassay is a mitogenic assayutilizing an IL-3 dependent clone of murine 32D cells that have beentransfected with human mpl receptor. This assay is described in greaterdetail in WO 95/26746. Cells are maintained in MEM medium containing 10%Fetal Clone II and 1 ng/ml mIL-3. Prior to sample addition, cells areprepared by rinsing twice with growth medium lacking mIL-3. An extendedtwelve point TPO standard curve is prepared, ranging from 33 to 39pg/ml. Four dilutions, estimated to fall within the linear portion ofthe standard curve, (100 to 125 pg/ml), are prepared for each sample andrun in triplicate. A volume of 100 μl of each dilution of sample orstandard is added to appropriate wells of a 96 well microtiter platecontaining 10,000 cells/well. After forty-four hours at 37° C. and 10%CO₂, MTS (a tetrazolium compound which is bioreduced by cells to aformazan) is added to each well. Approximately six hours later, theoptical density is read on a plate reader at 490 nm. A dose responsecurve (log TPO concentration vs. O.D.—Background) is generated andlinear regression analysis of points which fall in the linear portion ofthe standard curve is performed. Concentrations of unknown test samplesare determined using the resulting linear equation and a correction forthe dilution factor.

TMP tandem repeats with polyglycine linkers. Our design of sequentiallylinked TMP repeats was based on the assumption that a dimeric form ofTMP was required for its effective interaction with c-Mpl (the TPOreceptor) and that depending on how they were wound up against eachother in the receptor context, the two TMP molecules could be tetheredtogether in the C- to N-terminus configuration in a way that would notperturb the global dimeric conformation. Clearly, the success of thedesign of tandem linked repeats depends on proper selection of thelength and composition of the linker that joins the C- and N-termini ofthe two sequentially aligned TMP monomers. Since no structuralinformation of the TMP bound to c-Mpl was available, a series ofrepeated peptides with linkers composed of 0 to 10 and 14 glycineresidues (Table A) were synthesized. Glycine was chosen because of itssimplicity and flexibility, based on the rationale that a flexiblepolyglycine peptide chain might allow for the free folding of the twotethered TMP repeats into the required conformation, while other aminoacid sequences may adopt undesired secondary structures whose rigiditymight disrupt the correct packing of the repeated peptide in thereceptor context.

The resulting peptides are readily accessible by conventional solidphase peptide synthesis methods (Merrifield (1963), J. Amer. Chem. Soc.85: 2149) with either Fmoc or t-Boc chemistry. Unlike the synthesis ofthe C-terminally linked parallel dimer which required the use of anorthogonally protected lysine residue as the initial branch point tobuild the two peptide chains in a pseudosymmetrical way (Cwirla et al.(1997), Science 276: 1696–9), the synthesis of these tandem repeats wasa straightforward, stepwise assembly of the continuous peptide chainsfrom the C- to N-terminus. Since dimerization of TMP had a more dramaticeffect on the proliferative activity than binding affinity as shown forthe C-terminal dimer (Cwirla et al. (1997)), the synthetic peptides weretested directly for biological activity in a TPO-dependentcell-proliferation assay using an IL-3 dependent clone of murine 32Dcells transfected with the full-length c-Mpl (Palacios et al., Cell41:727 (1985)). As the test results showed, all the polyglycine linkedtandem repeats demonstrated >1000 fold increases in potency as comparedto the monomer, and were even more potent than the C-terminal dimer inthis cell proliferation assay. The absolute activity of the C-terminaldimer in our assay was lower than that of the native TPO protein, whichis different from the previously reported findings in which theC-terminal dimer was found to be as active as the natural ligand (Cwirlaet al. (1997)). This might be due to differences in the conditions usedin the two assays. Nevertheless, the difference in activity betweentandem (C terminal of first monomer linked to N terminal of secondmonomer) and C-terminal (C terminal of first monomer linked to Cterminal of second monomer; also referred to as parallel) dimers in thesame assay clearly demonstrated the superiority of tandem repeatstrategy over parallel peptide dimerization. It is interesting to notethat a wide range of length is tolerated by the linker. The optimallinker between tandem peptides with the selected TMP monomers apparentlyis composed of 8 glycines.

Other tandem repeats. Subsequent to this first series of TMP tandemrepeats, several other molecules were designed either with differentlinkers or containing modifications within the monomer itself. The firstof these molecules, peptide 13, has a linker composed of GPNG, asequence known to have a high propensity to form a β-turn-type secondarystructure. Although still about 100-fold more potent than the monomer,this peptide was found to be >10-fold less active than the equivalentGGGG-linked analog. Thus, introduction of a relatively rigid β-turn atthe linker region seemed to have caused a slight distortion of theoptimal agonist conformation in this short linker form.

The Trp9 in the TMP sequence is a highly conserved residue among theactive peptides isolated from random peptide libraries. There is also ahighly conserved Trp in the consensus sequences of EPO mimetic peptidesand this Trp residue was found to be involved in the formation of ahydrophobic core between the two EMPs and contributed to hydrophobicinteractions with the EPO receptor. Livnah et al. (1996), Science 273:464–71). By analogy, the Trp9 residue in TMP might have a similarfunction in dimerization of the peptide ligand, and as an attempt tomodulate and estimate the effects of noncovalent hydrophobic forcesexerted by the two indole rings, several analogs were made resultingfrom mutations at the Trp. So in peptide 14, the Trp residue wasreplaced in each of the two TMP monomers with a Cys, and anintramolecular disulfide bond was formed between the two cysteines byoxidation which was envisioned to mimic the hydrophobic interactionsbetween the two Trp residues in peptide dimerization. Peptide 15 is thereduced form of peptide 14. In peptide 16, the two Trp residues werereplaced by Ala. As the assay data show, all three analogs wereinactive. These data further demonstrated that Trp is critical for theactivity of the TPO mimetic peptide, not just for dimer formation.

The next two peptides (peptide 17a, and 18) each contain in their8-amino acid linker a Lys or Cys residue. These two compounds areprecursors to the two PEGylated peptides (peptide 19 and 20) in whichthe side chain of the Lys or Cys is modified by a PEG moiety. A PEGmoiety was introduced at the middle of a relatively long linker, so thatthe large PEG component (5 kDa) is far enough away from the criticalbinding sites in the peptide molecule. PEG is a known biocompatiblepolymer which is increasingly used as a covalent modifier to improve thepharmacokinetic profiles of peptide- and protein-based therapeutics.

A modular, solution-based method was devised for convenient PEGylationof synthetic or recombinant peptides. The method is based on the nowwell established chemoselective ligation strategy which utilizes thespecific reaction between a pair of mutually reactive functionalities.So, for pegylated peptide 19, the lysine side chain was preactivatedwith a bromoacetyl group to give peptide 17b to accommodate reactionwith a thiol-derivatized PEG. To do that, an orthogonal protectinggroup, Dde, was employed for the protection of the lysine E-amine. Oncethe whole peptide chain was assembled, the N-terminal amine wasreprotected with t-Boc. Dde was then removed to allow for thebromoacetylation. This strategy gave a high quality crude peptide whichwas easily purified using conventional reverse phase HPLC. Ligation ofthe peptide with the thiol-modified PEG took place in aqueous buffer atpH 8 and the reaction completed within 30 minutes. MALDI-MS analysis ofthe purified, pegylated material revealed a characteristic, bell-shapedspectrum with an increment of 44 Da between the adjacent peaks. ForPEG-peptide 20, a cysteine residue was placed in the linker region andits side chain thiol group would serve as an attachment site for amaleimide-containing PEG. Similar conditions were used for thepegylation of this peptide. As the assay data revealed, these twopegylated peptides had even higher in vitro bioactivity as compared totheir unpegylated counterparts.

Peptide 21 has in its 8-amino acid linker a potential glycosylationmotif, NGS. Since our exemplary tandem repeats are made up of naturalamino acids linked by peptide bonds, expression of such a molecule in anappropriate eukaryotic cell system should produce a glycopeptide withthe carbohydrate moiety added on the side chain carboxyamide of Asn.Glycosylation is a common post-translational modification process whichcan have many positive impacts on the biological activity of a givenprotein by increasing its aqueous solubility and in vivo stability. Asthe assay data show, incorporation of this glycosylation motif into thelinker maintained high bioactivity. The synthetic precursor of thepotential glycopeptide had in effect an activity comparable to that ofthe -(G)₈-linked analog. Once glycosylated, this peptide is expected tohave the same order of activity as the pegylated peptides, because ofthe similar chemophysical properties exhibited by a PEG and acarbohydrate moiety.

The last peptide is a dimer of a tandem repeat. It was prepared byoxidizing peptide 18, which formed an intermolecular disulfide bondbetween the two cysteine residues located at the linker. This peptidewas designed to address the possibility that TMP was active as atetramer. The assay data showed that this peptide was not more activethan an average tandem repeat on an adjusted molar basis, whichindirectly supports the idea that the active form of TMP is indeed adimer, otherwise dimerization of a tandem repeat would have a furtherimpact on the bioactivity.

In order to confirm the in vitro data in animals, one pegylated TMPtandem repeat (compound 20 in Table A) was delivered subcutaneously tonormal mice via osmotic pumps. Time and dose-dependent increases wereseen in platelet numbers for the duration of treatment. Peak plateletlevels over 4-fold baseline were seen on day 8. A dose of 10 μg/kg/dayof the pegylated TMP repeat produced a similar response to rHuMGDF(non-pegylated) at 100 μg/kg/day delivered by the same route.

TABLE A TPO-mimetic Peptides Peptide SEQ ID Relative No. Compound NO:Potency TPO ++++ TMP monomer  13 + TMP C—C dimer +++− TMP-(G)_(n)-TMP: 1 n = 0 341 ++++−  2 n = 1 342 ++++  3 n = 2 343 ++++  4 n = 3 344 ++++ 5 n = 4 345 ++++  6 n = 5 346 ++++  7 n = 6 347 ++++  8 n = 7 348 ++++ 9 n = 8 349 ++++− 10 n = 9 350 ++++ 11 n = 10 351 ++++ 12 n = 14 352++++ 13 TMP-GPNG-TMP 353 +++ 14

(cyclic) 354 − 15 IEGPTLRQCLAARA-GGGGGGGG- 355 − IEGPTLRQCLAARA (linear)16 IEGPTLRQALAARA-GGGGGGGG- 356 − IEGPTLRQALAARA 17a TMP-GGGKGGGG-TMP357 ++++ 17b TMP-GGGK(BrAc)GGGG-TMP 358 ND 18 TMP-GGGCGGGG-TMP 359 ++++19 TMP-GGGK(PEG)GGGG-TMP 360 +++++ 20 TMP-GGGC(PEG)GGGG-TMP 361 +++++ 21TMP-GGGN*GSGG-TMP 362 ++++ 22

363 363 ++++

Discussion. It is well accepted that MGDF acts in a way similar to hGH,i.e., one molecule of the protein ligand binds two molecules of thereceptor for its activation. Wells et al.(1996), Ann. Rev. Biochem. 65:609–34. Now, this interaction is mimicked by the action of a muchsmaller peptide, TMP. However, the present studies suggest that thismimicry requires the concerted action of two TMP molecules, as covalentdimerization of TMP in either a C—C parallel or C—N sequential fashionincreased the in vitro biological potency of the original monomer by afactor of greater than 10³. The relatively low biopotency of the monomeris probably due to inefficient formation of the noncovalent dimer. Apreformed covalent repeat has the ability to eliminate the entropybarrier for the formation of a noncovalent dimer which is exclusivelydriven by weak, noncovalent interactions between two molecules of thesmall, 14-residue peptide.

It is intriguing that this tandem repeat approach had a similar effecton enhancing bioactivity as the reported C—C dimerization is intriguing.These two strategies brought about two very different molecularconfigurations. The C—C dimer is a quasi-symmetrical molecule, while thetandem repeats have no such symmetry in their linear structures. Despitethis difference in their primary structures, these two types ofmolecules appeared able to fold effectively into a similar biologicallyactive conformation and cause the dimerization and activation of c-Mpl.These experimental observations provide a number of insights into howthe two TMP molecules may interact with one another in binding to c-Mpl.First, the two C-termini of the two bound TMP molecules must be inrelatively close proximity with each other, as suggested by data on theC-terminal dimer. Second, the respective N- and C-termini of the two TMPmolecules in the receptor complex must also be very closely aligned witheach other, such that they can be directly tethered together with asingle peptide bond to realize the near maximum activity-enhancingeffect brought about by the tandem repeat strategy. Insertion of one ormore (up to 14) glycine residues at the junction did not increase (ordecrease) significantly the activity any further. This may be due to thefact that a flexible polyglycine peptide chain is able to loop outeasily from the junction without causing any significant changes in theoverall conformation. This flexibility seems to provide the freedom oforientation for the TMP peptide chains to fold into the requiredconformation in interacting with the receptor and validate it as a siteof modification. Indirect evidence supporting this came from the studyon peptide 13, in which a much more rigid b-turn-forming sequence as thelinker apparently forced a deviation of the backbone alignment aroundthe linker which might have resulted in a slight distortion of theoptimal conformation, thus resulting in a moderate (10-fold) decrease inactivity as compared with the analogous compound with a 4-Gly linker.Third, Trp9 in TMP plays a similar role as Trp13 in EMP, which isinvolved not only in peptide:peptide interaction for the formation ofdimers but also is important for contributing hydrophobic forces inpeptide:receptor interaction. Results obtained with the W to C mutantanalog, peptide 14, suggest that a covalent disulfide linkage is notsufficient to approximate the hydrophobic interactions provided by theTrp pair and that, being a short linkage, it might bring the two TMPmonomers too close, therefore perturbing the overall conformation of theoptimal dimeric structure.

An analysis of the possible secondary structure of the TMP peptide canprovide further understanding on the interaction between TMP and c-Mpl.This can be facilitated by making reference to the reported structure ofthe EPO mimetic peptide. Livnah et al. (1996), Science 273:464–75 Thereceptor-bound EMP has a b-hairpin structure with a b-turn formed by thehighly consensus Gly-Pro-Leu-Thr at the center of its sequence. Insteadof GPLT, TMP has a highly selected GPTL sequence which is likely to forma similar turn. However, this turn-like motif is located near theN-terminal part in TMP. Secondary structure prediction using Chau-Fasmanmethod suggests that the C-terminal half of the peptide has a tendencyto adopt a helical conformation. Together with the highly conserved Trpat position 9, this C-terminal helix may contribute to the stabilizationof the dimeric structure. It is interesting to note that most of ourtandem repeats are more potent than the C-terminal parallel dimer.Tandem repeats seem to give the molecule a better fit conformation thandoes the C—C parallel dimerization. The seemingly asymmetric feature ofa tandem repeat might have brought it closer to the natural ligandwhich, as an asymmetric molecule, uses two different sites to bind twoidentical receptor molecules.

Introduction of a PEG moiety was envisaged to enhance the in vivoactivity of the modified peptide by providing it a protection againstproteolytic degradation and by slowing down its clearance through renalfiltration. It was unexpected that pegylation could further increase thein vitro bioactivity of a tandem repeated TMP peptide in the cell-basedproliferation assay.

Example 2 Fc-TMP Fusions

TMPs (and EMPs as described in Example 3) were expressed in eithermonomeric or dimeric form as either N-terminal or C-terminal fusions tothe Fc region of human IgG1. In all cases, the expression constructutilized the luxPR promoter promoter in the plasmid expression vectorpAMG21.

Fc-TMP. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a monomer of the TPO-mimetic peptide was constructed usingstandard PCR technology. Templates for PCR reactions were the pFc-A3vector and a synthetic TMP gene. The synthetic gene was constructed fromthe 3 overlapping oligonucleotides (SEQ ID NOS: 364, 365, and 366,respectively) shown below:

1842-97 AAA AAA GGA TCC TCG AGA TTA AGC ACG AGC AGC CAG CCA CTG ACg CAGAGT CGG ACC 1842-98 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG ACT CTG CGT1842-99 CAG TGG CTG GCT GCT CGT GCT TAA TCT CGA GGA TCC TTT TTTThese oligonucleotides were annealed to form the duplex encoding anamino acid sequence (SEQ ID NOS: 367 and 368, respectively) shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1---------+---------+---------+---------+---------+---------+ 60                        CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA aK  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A -TAATCTCGAGGATCCTTTTTT 61 ---------+---------+- 81 ATTAGAGCTCCTAGGAAAAAAa *This duplex was amplified in a PCR reaction using 1842–98 and 1842–97 asthe sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction withpFc-A3 using the primers shown below (SEQ ID NOS: 369 and 370):

1216-52 AAC ATA AGT ACC TGT AGG ATC G 1830-51 TTCGATACCA CCACCTCCACCTTTACCCGG AGACAGGGAG AGGCTCTTCTGCThe oligonucleotides 1830–51 and 1842–98 contain an overlap of 24nucleotides, allowing the two genes to be fused together in the correctreading frame by combining the above PCR products in a third reactionusing the outside primers, 1216–52 and 1842–97.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #3728.

The nucleotide and amino acid sequences (SEQ ID NOS: 5 and 6) of thefusion protein are shown in FIG. 7.

Fc-TMP-TMP. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a dimer of the TPO-mimetic peptide was constructed usingstandard PCR technology. Templates for PCR reactions were the pFc-A3vector and a synthetic TMP-TMP gene. The synthetic gene was constructedfrom the 4 overlapping oligonucleotides (SEQ ID NOS: 371 to 374,respectively) shown below:

1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG ACT CTG CGT CAG TGG CTGGCT GCT CGT GCT 1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAG CCA CTG ACGCAG ACT CGG ACC 1830-54 GGT GGT GGA GGT GGC GGC GGA GGT ATT GAG GGC CCAACC CTT CGC CAA TGG CTT GCA GCA CGC GCA 1830-55 AAA AAA AGG ATC CTC GAGATT ATG CGC GTG CTG CAA GCC ATT GGC GAA GGG TTG GGC CCT CAA TAC CTC CGCCGC CThe 4 oligonucleotides were annealed to form the duplex encoding anamino acid sequence (SEQ ID NOS: 375 and 376, respectively) shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1---------+---------+---------+---------+---------+---------+ 60                        CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA aK  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A -GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA 61---------+---------+---------+---------+---------+---------+ 120CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT aG  G  G  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A - CGCGCA 121---------------------------148 GCGCGTATTAGAGCTCCTAGGAAAAAAA a R  A   *-This duplex was amplified in a PCR reaction using 1830–52 and 1830–55 asthe sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction withpFc-A3 using the primers 1216–52 and 1830–51 as described above forFc-TMP. The full length fusion gene was obtained from a third PCRreaction using the outside primers 1216–52 and 1830–55.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described in example 1. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #3727.

The nucleotide and amino acid sequences (SEQ ID NOS: 7 and 8) of thefusion protein are shown in FIG. 8.

TMP-TMP-Fc. A DNA sequence coding for a tandem repeat of the TPO-mimeticpeptide fused in-frame to the Fc region of human IgG1 was constructedusing standard PCR technology. Templates for PCR reactions were theEMP-Fc plasmid from strain #3688 (see Example 3) and a synthetic geneencoding the TMP dimer. The synthetic gene for the tandem repeat wasconstructed from the 7 overlapping oligonucleotides shown below (SEQ IDNOS: 377 to 383, respectively):

1885-52 TTT TTT CAT ATG ATC GAA GGT CCG ACT CTG CGT CAG TGG 1885-53 AGCACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC TTC GAT CAT ATG 1885-54 CTGGCT GCT CGT GCT GGT GGA GGC GGT GGG GAC AAA ACT CAC ACA 1885-55 CTG GCTGCT CGT GCT GGC GGT GGT GGC GGA GGG GGT GGC ATT GAG GGC CCA 1885-56 AAGCCA TTG GCG AAG GGT TGG GCC CTC AAT GCC ACC CCC TCC GCC ACC ACC GCC1885-57 ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA GGG GGA GGC GGT GGG GACAAA ACT 1885-58 CCC ACC GCC TCC CCC TGC GCG TGC TGCThese oligonucleotides were annealed to form the duplex shown encodingan amino acid sequence shown below (SEQ ID NOS 384 and 385):

TTTTTTCATATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGT 1---------+---------+---------+---------+---------+---------+ 60      GTATACTAGCTTCCAGGCTGAGACGCAGTCACCGACCGACGAGCACGACCGCCA a          M  I  E  G  P  T  L  R  Q  W  L  A  A  R  A  G  G -GGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTGGCTGCTCGTGCT 61---------+---------+---------+---------+---------+---------+ 120CCACCGCCTCCCCCACCGTAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGCGCGT aG  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A -GGTGGAGGCGGTGGGGACAAAACTCTGGCTGCTCGTGCTGGTGGAGGCGGTGGGGACAAA 121---------+---------+---------+---------+---------+---------+ 180CCCCCTCCGCCACCC aG  G  G  G  G  D  K  T  L  A  A  R  A  G  G  G  G  G  D  K - ACTCACACA181 --------- 189 a T  H  T  -This duplex was amplified in a PCR reaction using 1885–52 and 1885–58 asthe sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction with DNAfrom the EMP-Fc fusion strain #3688 (see Example 3) using the primers1885–54 and 1200–54. The full length fusion gene was obtained from athird PCR reaction using the outside primers 1885–52 and 1200–54.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for Fc-EMP herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #3798.

The nucelotide and amino acid sequences (SEQ ID NOS: 9 and 10) of thefusion protein are shown in FIG. 9.

TMP-Fc. A DNA sequence coding for a monomer of the TPO-mimetic peptidefused in-frame to the Fc region of human IgG1 was obtained fortuitouslyin the ligation in TMP-TMP-Fc, presumably due to the ability of primer1885–54 to anneal to 1885–53 as well as to 1885–58. A single clonehaving the correct nucleotide sequence for the TMP-Fc construct wasselected and designated Amgen strain #3788.

The nucleotide and amino acid sequences (SEQ ID NOS: 11 and 12) of thefusion protein are shown in FIG. 10.

Expression in E. coli. Cultures of each of the pAMG21-Fc-fusionconstructs in E. coli GM221 were grown at 37° C. in Luria Broth mediumcontaining 50 mg/ml kanamycin. Induction of gene product expression fromthe luxPR promoter was achieved following the addition of the syntheticautoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture mediato a final concentration of 20 ng/ml. Cultures were incubated at 37° C.for a further 3 hours. After 3 hours, the bacterial cultures wereexamined by microscopy for the presence of inclusion bodies and werethen collected by centrifugation. Refractile inclusion bodies wereobserved in induced cultures indicating that the Fc-fusions were mostlikely produced in the insoluble fraction in E. coli. Cell pellets werelysed directly by resuspension in Laemmli sample buffer containing 10%b-mercaptoethanol and were analyzed by SDS-PAGE. In each case, anintense coomassie-stained band of the appropriate molecular weight wasobserved on an SDS-PAGE gel.

pAMG21. The expression plasmid pAMG21 can be derived from the Amgenexpression vector pCFM1656 (ATCC #69576) which in turn be derived fromthe Amgen expression vector system described in U.S. Pat. No. 4,710,473.The pCFM1656 plasmid can be derived from the described pCFM836 plasmid(U.S. Pat. No. 4,710,473) by:

-   -   (a) destroying the two endogenous NdeI restriction sites by end        filling with T4 polymerase enzyme followed by blunt end        ligation;    -   (b) replacing the DNA sequence between the unique AatII and ClaI        restriction sites containing the synthetic P_(L) promoter with a        similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473)        containing the PL promoter (see SEQ ID NO: 386 below); and    -   (c) substituting the small DNA sequence between the unique ClaI        and KpnI restriction sites with the oligonucleotide having the        sequence of SEQ ID NO: 388.

AatII 5′ CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- SEQ IDNO:386 3′ TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA--AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA--TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT--TACCACTGGCGGTGATACTGAGCACAT 3′ -ATGGTGACCGCCACTATGACTCGTGTAGC 5′                            ClaI5′ CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′ SEQ IDNO:387 3′ TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′   ClaI                                               KpnIThe expression plasmid pAMG21 can then be derived from pCFM1656 bymaking a series of site-directed base changes by PCR overlapping oligomutagenesis and DNA sequence substitutions. Starting with the BglII site(plasmid bp # 180) immediately 5′ to the plasmid replication promoterP_(copB) and proceeding toward the plasmid replication genes, the basepair changes are as shown in Table B below.

TABLE B Base pair changes resulting in pAMG21 pAMG21 bp # bp in pCFM1656bp changed to in pAMG21 # 204 T/A C/G # 428 A/T G/C # 509 G/C A/T # 617— insert two G/C bp # 679 G/C T/A # 980 T/A C/G # 994 G/C A/T # 1004 A/TC/G # 1007 C/G T/A # 1028 A/T T/A # 1047 C/G T/A # 1178 G/C T/A # 1466G/C T/A # 2028 G/C bp deletion # 2187 C/G T/A # 2480 A/T T/A # 2499–2502AGTG GTCA TCAC CAGT # 2642 TCCGAGC 7 bp deletion AGGCTCG # 3435 G/C A/T# 3446 G/C A/T # 3643 A/T T/A

The DNA sequence between the unique AatII (position #4364 in pCFM1656)and SacII (position #4585 in pCFM1656) restriction sites is substitutedwith the DNA sequence (SEQ ID NO: 23) shown in FIGS. 17A and 17B. Duringthe ligation of the sticky ends of this substitution DNA sequence, theoutside AatII and SacII sites are destroyed. There are unique AatII andSacII sites in the substituted DNA.

GM221 (Amgen #2596). The Amgen host strain #2596 is an E. coli K-12strain derived from Amgen strain #393. It has been modified to containboth the temperature sensitive lambda repressor cI857s7 in the early ebgregion and the lacI^(Q) repressor in the late ebg region (68 minutes).The presence of these two repressor genes allows the use of this hostwith a variety of expression systems, however both of these repressorsare irrelevant to the expression from luxP_(R). The untransformed hosthas no antibiotic resistances.

The ribosome binding site of the cI857s7 gene has been modified toinclude an enhanced RBS. It has been inserted into the ebg operonbetween nucleotide position 1170 and 1411 as numbered in Genbankaccession number M64441Gb_Ba with deletion of the intervening ebgsequence. The sequence of the insert is shown below with lower caseletters representing the ebg sequences flanking the insert shown below

(SEQ ID NO:388)ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGTAAACTAGTCAACACGCACGGTGTTAGATATTTATCCCTTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAGACTAGTGGATCCACTAGTgtttctgccc

The construct was delivered to the chromosome using a recombinant phagecalled MMebg-cI857s7 enhanced RBS #4 into F'tet/393. After recombinationand resolution only the chromosomal insert described above remains inthe cell. It was renamed F'tet/GM101. F'tet/GM101 was then modified bythe delivery of a lacI^(Q) construct into the ebg operon betweennucleotide position 2493 and 2937 as numbered in the Genbank accessionnumber M64441Gb_Ba with the deletion of the intervening ebg sequence.The sequence of the insert is shown below with the lower case lettersrepresenting the ebg sequences flanking the insert (SEQ ID NO: 389)shown below:

ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTCGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga

The construct was delivered to the chromosome using a recombinant phagecalled AGebg-LacIQ#5 into F'tet/GM101. After recombination andresolution only the chromosomal insert described above remains in thecell. It was renamed F'tet/GM221. The F'tet episome was cured from thestrain using acridine orange at a concentration of 25 μg/ml in LB. Thecured strain was identified as tetracyline sensitive and was stored asGM221.

Expression. Cultures of pAMG21-Fc-TMP-TMP in E. coli GM221 in LuriaBroth medium containing 50 μg/ml kanamycin were incubated at 37° C.prior to induction. Induction of Fc-TMP-TMP gene product expression fromthe luxPR promoter was achieved following the addition of the syntheticautoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture mediato a final concentration of 20 ng/ml and cultures were incubated at 37°C. for a further 3 hours. After 3 hours, the bacterial cultures wereexamined by microscopy for the presence of inclusion bodies and werethen collected by centrifugation. Refractile inclusion bodies wereobserved in induced cultures indicating that the Fc-TMP-TMP was mostlikely produced in the insoluble fraction in E. coli. Cell pellets werelysed directly by resuspension in Laemmli sample buffer containing 10%•-mercaptoethanol and were analyzed by SDS-PAGE. An intense Coomassiestained band of approximately 30 kDa was observed on an SDS-PAGE gel.The expected gene product would be 269 amino acids in length and have anexpected molecular weight of about 29.5 kDa. Fermentation was alsocarried out under standard batch conditions at the 10 L scale, resultingin similar expression levels of the Fc-TMP-TMP to those obtained atbench scale.

Purification of Fc-TMP-TMP. Cells are broken in water (1/10) by highpressure homogenization (2 passes at 14,000 PSI) and inclusion bodiesare harvested by centrifugation (4200 RPM in J-6B for 1 hour). Inclusionbodies are solubilized in 6M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for1 hour at a 1/10 ratio. The solubilized mixture is diluted 20 times into2M urea, 50 mM tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixtureis stirred overnight in the cold and then concentrated about 10 fold byultafiltration. It is then diluted 3 fold with 10 mM Tris, 1.5M urea, pH9. The pH of this mixture is then adjusted to pH 5 with acetic acid. Theprecipitate is removed by centrifugation and the supernatant is loadedonto a SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mMNaCl, pH 5(10 mg/ml protein load, room temperature). The protein iseluted off using a 20 column volume gradient in the same buffer rangingfrom 100 mM NaCl to 500 mM NaCl. The pool from the column is diluted 3fold and loaded onto a SP-Sepharose HP column in 20 mM NaAc, 150 mMNaCl, pH 5(10 mg/ml protein load, room temperature). The protein iseluted off using a 20 column volume gradient in the same buffer rangingfrom 150 mM NaCl to 400 mM NaCl. The peak is pooled and filtered.

Characterization of Fc-TMP activity. The following is a summary of invivo data in mice with various compounds of this invention.

Mice: Normal female BDF1 approximately 10–12 weeks of age.

Bleed schedule: Ten mice per group treated on day 0, two groups started4 days apart for a total of 20 mice per group. Five mice bled at eachtime point, mice were bled a minimum of three times a week. Mice wereanesthetized with isoflurane and a total volume of 140–160 μl of bloodwas obtained by puncture of the orbital sinus. Blood was counted on aTechnicon H1E blood analyzer running software for murine blood.Parameters measured were white blood cells, red blood cells, hematocrit,hemoglobin, platelets, neutrophils.

Treatments: Mice were either injected subcutaneously for a bolustreatment or implanted with 7-day micro-osmotic pumps for continuousdelivery. Subcutaneous injections were delivered in a volume of 0.2 ml.Osmotic pumps were inserted into a subcutaneous incision made in theskin between the scapulae of anesthetized mice. Compounds were dilutedin PBS with 0.1% BSA. All experiments included one control group,labeled “carrier” that were treated with this diluent only. Theconcentration of the test articles in the pumps was adjusted so that thecalibrated flow rate from the pumps gave the treatment levels indicatedin the graphs.

Compounds: A dose titration of the compound was delivered to mice in 7day micro-osmotic pumps. Mice were treated with various compounds at asingle dose of 100 μg/kg in 7 day osmotic pumps. Some of the samecompounds were then given to mice as a single bolus injection.

Activity test results: The results of the activity experiments are shownin FIGS. 11 and 12. In dose response assays using 7-day micro-osmoticpumps, the maximum effect was seen with the compound of SEQ ID NO: 18was at 100 μg/kg/day; the 10 μg/kg/day dose was about 50% maximallyactive and 1 μg/kg/day was the lowest dose at which activity could beseen in this assay system. The compound at 10 μg/kg/day dose was aboutequally active as 100 μg/kg/day unpegylated rHu-MGDF in the sameexperiment.

Example 3 Fc-EMP Fusions

Fc-EMP. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a monomer of the EPO-mimetic peptide was constructed usingstandard PCR technology. Templates for PCR reactions were a vectorcontaining the Fc sequence (pFc-A3, described in Internationalapplication WO 97/23614, published Jul. 3, 1997) and a synthetic geneencoding EPO monomer. The synthetic gene for the monomer was constructedfrom the 4 overlapping oligonucleotides (SEQ ID NOS: 390 to 393,respectively) shown below:

1798-2 TAT GAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTC TTG CCA CTT CGGCCC GCT GAC TTG G 1798-3 CGG TTT GCA AAC CCA AGT CAG CGG GCC GAA GTG GCAAGA GTA AGT ACC TCC ACC ACC ACC TCC ACC TTT CAT 1798-4 GTT TGC AAA CCGCAG GGT GGC GGC GGC GGC GGC GGT GGT ACC TAT TCC TGT CAT TTT 1798-5 CCAGGT CAG CGG GCC AAA ATG ACA GGA ATA GGT ACC ACC GCC GCC GCC GCC GCC ACCCTGThe 4 oligonucleotides were annealed to form the duplex encoding anamino acid sequence (SEQ ID NOS: 394 and 395, respectively) shown below:

TATGAAAGGTGGAGGTGGTGGTGGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTG 1---------+---------+---------+---------+---------+---------+ 60TACTTTCCACCTCCACCACCACCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAAC b M  K  G  G  G  G  G  G  G  T  Y  S  C  H  F  G  P  L  T  W -GGTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTT 61---------+---------+---------+---------+---------+---------+---------+--133CCAAACGTTTGGCGTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCb  V  C  K  P  Q  G  G  G  G  G  G  G  G  T  Y  S  C  H  F  -

duplex was amplified in a PCR reaction using

1798-18 GCA GAA GAG CCT CTC CCT GTC TCC GGG TAA AGG TGG AGG TGG TGG TGGAGG TAC TTA CTC T and 1798-19 CTA ATT GGA TCC ACG AGA TTA ACC ACC CTGCGG TTT GCA Aas the sense and antisense primers (SEQ ID NOS: 396 and 397,respectively).

The Fc portion of the molecule was generated in a PCR reaction withpFc-A3 using the primers

1216-52 AAC ATA AGT ACC TGT AGG ATC G 1798-17 AGA GTA ACT ACC TCC ACCACC ACC TCC ACC TTT ACC CGG AGA CAG GGA GAG GCT CTT CTG Cwhich are SEQ ID NOS: 369 and 399, respectively. The oligonucleotides1798-17 and 1798-18 contain an overlap of 61 nucleotides, allowing thetwo genes to be fused together in the correct reading frame by combiningthe above PCR products in a third reaction using the outside primers,1216-52 and 1798-19.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 (described below), also digested with XbaI and BamHI.Ligated DNA was transformed into competent host cells of E. coli strain2596 (GM221, described herein). Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #3718.

The nucleotide and amino acid sequence of the resulting fusion protein(SEQ ID NOS: 15 and 16) are shown in FIG. 13.

EMP-Fc. A DNA sequence coding for a monomer of the EPO-mimetic peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. Templates for PCR reactions were the pFC-A3avector and a synthetic gene encoding EPO monomer. The synthetic gene forthe monomer was constructed from the 4 overlapping oligonucleotides1798-4 and 1798-5 (above) and 1798-6 and 1798-7 (SEQ ID NOS: 400 and401, respectively) shown below:

1798-6 CCC CCG CTG ACC TGG GTA TGT AAC CCA CAA GGG GGT GGG GGA GCC GGGGGG TAA TCT CCA C 1798-7 CAT CCT CGA GAT TAC CCC CCG CCT CCC CCA CCC CCTTGT GGC TTA CAT ACThe 4 oligonucleotides were annealed to form the duplex encoding anamino acid sequence (SEQ ID NOS: 402 and 403, respectively) shown below:

GTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGC 1---------+---------+---------+---------+---------+---------+ 60            GTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCG AV  C  K  P  Q  G  G  G  G  G  G  G  G  T  Y  S  C  H  F  G -CCGCTGACCTGGGTATGTAAGCCACAAGGGGGTGGGGGAGGCGGGGGGTAATCTCGAG 61---------+---------+---------+---------+---------+---------+- 122GGCGACTGGACCCATACATTCGGTGTTCCCCCACCCCCTCCGCCCCCCATTAGAGCTCCTAG AP  L  T  W  V  C  K  P  Q  G  G  G  G  G  G  G  *

This duplex was amplified in a PCR reaction using

1798-21 TTA TTT CAT ATG AAA GGT GGT AAC TAT TCC TGT CAT TTT and 1798-22TGG ACA TGT GTG AGT TTT GTC CCC CCC GCC TCC CCC ACC CCC Tas the sense and antisense primers (SEQ ID NOS: 404 and 405,respectively).

The Fc portion of the molecule was generated in a PCR reaction withpFc-A3 using the primers

1798-23 AGG GGG TGG GGG AGG CGG GGG GGA CAA AAC TCA CAC ATG TCC A and1200-54 GTT ATT GCT CAG CGG TGG CAwhich are SEQ ID NOS: 406 and 407, respectively. The oligonucleotides1798-22 and 1798-23 contain an overlap of 43 nucleotides, allowing thetwo genes to be fused together in the correct reading frame by combiningthe above PCR products in a third reaction using the outside primers,1787-21 and 1200-54.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described above. Clones were screened for the ability to produce therecombinant protein product and to possess the gene fusion having thecorrect nucleotide sequence. A single such clone was selected anddesignated Amgen strain #3688.

The nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18) of theresulting fusion protein are shown in FIG. 14.

EMP-EMP-Fc. A DNA sequence coding for a dimer of the EPO-mimetic peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. Templates for PCR reactions were the EMP-Fcplasmid from strain #3688 above and a synthetic gene encoding the EPOdimer. The synthetic gene for the dimer was constructed from the 8overlapping oligonucleotides (SEQ ID NOS:408 to 415, respectively) shownbelow:

1869-23 TTT TTT ATC GAT TTG ATT CTA GAT TTG AGT TTT AAC TTT TAG AAG GAGGAA TAA AAT ATG 1869-48 TAA AAG TTA AAA CTC AAA TCT AGA ATC AAA TCG ATAAAA AA 1871-72 GGA GGT ACT TAC TCT TGC CAC TTC GGC CCG CTG ACT TGG GTTTGC AAA CCG 1871-73 AGT CAG CGG GCC GAA GTG GCA AGA GTA AGT ACC TCC CATATT TTA TTC CTC CTT C 1871-74 CAG GGT GGC GGC GGC GGC GGC GGT GGT ACCTAT TCC TGT CAT TTT GGC CCG CTG ACC TGG 1871-75 AAA ATG ACA GGA ATA GGTACC ACC GCC GCC GCC GCC GCC ACC CTG CGG TTT GCA AAC CCA 1871-78 GTA TGTAAG CCA CAA GGG GGT GGG GGA GGC GGG GGG GAC AAA ACT CAC ACA TGT CCA1871-79 AGT TTT GTC CCC CCC GCC TCC CCC ACC CCC TTG TGG CTT ACA TAC CCAGGT CAG CGG GCCThe 8 oligonucleotides were annealed to form the duplex encoding anamino acid sequence (SEQ ID NOS: 416 and 417, respectively) shown below:

TTTTTTATCGATTTGATTCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAAAATATG 1---------+---------+---------+---------+---------+---------+ 60AAAAAATAGCTAAACTAAGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTTTATAC a                                                          M -GGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTGGGTTTGCAAACCGCAGGGTGGC 61---------+---------+---------+---------+---------+---------+ 120CCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAACCCAAACGTTTGGCGTCCCACCG aG  G  T  Y  S  C  H  F  G  P  L  T  W  V  C  K  P  Q  G  G -GGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGCCCGCTGACCTGGGTATGTAAG 121---------+---------+---------+---------+---------+---------+ 180CCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCCATACATTC aG  G  G  G  G  G  T  Y  S  C  H  F  G  P  L  T  W  V  C  K -CCACAAGGGGGTGGGGGAGGCGGGGGGGACAAAACTCACACATGTCCA 181---------+---------+---------+---------+-------- 228GGTGTTCCCCCACCCCCTCCGCCCCCCCTGTTTTGA aP  Q  G  G  G  G  G  G  G  D  K  T  H  T  C  P  -

This duplex was amplified in a PCR reaction using 1869-23 and 1871-79(shown above) as the sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction withstrain 3688 DNA using the primers 1798-23 and 1200-54 (shown above).

The oligonucleotides 1871-79 and 1798-23 contain an overlap of 31nucleotides, allowing the two genes to be fused together in the correctreading frame by combining the above PCR products in a third reactionusing the outside primers, 1869-23 and 1200-54.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for Fc-EMP. Clones were screened for ability to produce therecombinant protein product and possession of the gene fusion having thecorrect nucleotide sequence. A single such clone was selected anddesignated Amgen strain #3813.

The nucleotide and amino acid sequences (SEQ ID NOS: 19 and 20,respectively) of the resulting fusion protein are shown in FIG. 15.There is a silent mutation at position 145 (A to G, shown in boldface)such that the final construct has a different nucleotide sequence thanthe oligonucleotide 1871-72 from which it was derived.

Fc-EMP-EMP. A DNA sequence coding for the Fc region of human IgG1 fusedin-frame to a dimer of the EPO-mimetic peptide was constructed usingstandard PCR technology. Templates for PCR reactions were the plasmidsfrom strains 3688 and 3813 above.

The Fc portion of the molecule was generated in a PCR reaction withstrain 3688 DNA using the primers 1216-52 and 1798-17 (shown above). TheEMP dimer portion of the molecule was the product of a second PCRreaction with strain 3813 DNA using the primers 1798-18 (also shownabove) and SEQ ID NO: 418, shown below:1798-20 CTA ATT GGA TCC TCG AGA TTA ACC CCC TTG TGG CTT ACAT

The oligonucleotides 1798-17 and 1798-18 contain an overlap of 61nucleotides, allowing the two genes to be fused together in the correctreading frame by combining the above PCR products in a third reactionusing the outside primers, 1216-52 and 1798-20.

The final PCR gene product (the full length fusion gene) was digestedwith restriction endonucleases XbaI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for Fc-EMP. Clones were screened for the ability to producethe recombinant protein product and to possess the gene fusion havingthe correct nucleotide sequence. A single such clone was selected anddesignated Amgen strain #3822.

The nucleotide and amino acid sequences (SEQ ID NOS: 21 and 22,respectively) of the fusion protein are shown in FIG. 16.

Characterization of Fc-EMP activity. Characterization was carried out invivo as follows.

Mice: Normal female BDF1 approximately 10–12 weeks of age.

Bleed schedule: Ten mice per group treated on day 0, two groups started4 days apart for a total of 20 mice per group. Five mice bled at eachtime point, mice were bled a maximum of three times a week. Mice wereanesthetized with isoflurane and a total volume of 140–160 ml of bloodwas obtained by puncture of the orbital sinus. Blood was counted on aTechnicon H1E blood analyzer running software for murine blood.Parameters measured were WBC, RBC, HCT, HGB, PLT, NEUT, LYMPH.

Treatments: Mice were either injected subcutaneously for a bolustreatment or implanted with 7 day micro-osmotic pumps for continuousdelivery. Subcutaneous injections were delivered in a volume of 0.2 ml.Osmotic pumps were inserted into a subcutaneous incision made in theskin between the scapulae of anesthetized mice. Compounds were dilutedin PBS with 0.1% BSA. All experiments included one control group,labeled “carrier” that were treated with this diluent only. Theconcentration of the test articles in the pumps was adjusted so that thecalibrated flow rate from the pumps gave the treatment levels indicatedin the graphs.

Experiments: Various Fc-conjugated EPO mimetic peptides (EMPs) weredelivered to mice as a single bolus injection at a dose of 100 μg/kg.Fc-EMPs were delivered to mice in 7-day micro-osmotic pumps. The pumpswere not replaced at the end of 7 days. Mice were bled until day 51 whenHGB and HCT returned to baseline levels.

Example 4 TNF-α Inhibitors

Fc-TNF-α inhibitors. A DNA sequence coding for the Fc region of humanIgG1 fused in-frame to a monomer of the TNF-α inhibitory peptide wasconstructed using standard PCR technology. The Fc and 5 glycine linkerportion of the molecule was generated in a PCR reaction with DNA fromthe Fc-EMP fusion strain #3718 (see Example 3) using the sense primer1216-52 and the antisense primer 2295-89 (SEQ ID NOS: 369 and 1112,respectively). The nucleotides encoding the TNF-α inhibitory peptidewere provided by the PCR primer 2295-89 shown below:

1216-52 AAC ATA AGT ACC TGT AGG ATC G 2295-89 CCG CGG ATC CAT TAC GGACGG TGA CCC AGA GAG GTG TTT TTG TAG TGC GGC AGG AAG TCA CCA CCA CCT CCACCT TTA CCCThe oligonucleotide 2295-89 overlaps the glycine linker and Fc portionof the template by 22 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4544.

The nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) ofthe fusion protein are shown in FIGS. 19A and 19B.

TNF-α inhibitor-Fc. A DNA sequence coding for a TNF-α inhibitory peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. The template for the PCR reaction was a plasmidcontaining an unrelated peptide fused via a five glycine linker to Fc.The nucleotides encoding the TNF-α inhibitory peptide were provided bythe sense PCR primer 2295-88, with primer 1200-54 serving as theantisense primer (SEQ ID NOS: 1117 and 407, respectively). The primersequences are shown below:

2295-88 GAA TAA CAT ATG GAC TTC CTG CCG CAC TAC AAA AAC ACC TCT CTG GGTCAC CGT CCG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT GCT CAG CGGTGG CAThe oligonucleotide 2295-88 overlaps the glycine linker and Fc portionof the template by 24 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4543.

The nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) ofthe fusion protein are shown in FIGS. 20A and 20B.

Expression in E. coli. Cultures of each of the pAMG21-Fc-fusionconstructs in E. coli GM221 were grown at 37° C. in Luria Broth mediumcontaining 50 mg/ml kanamycin. Induction of gene product expression fromthe luxPR promoter was achieved following the addition of the syntheticautoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture mediato a final concentration of 20 ng/ml. Cultures were incubated at 37° C.for a further 3 hours. After 3 hours, the bacterial cultures wereexamined by microscopy for the presence of inclusion bodies and werethen collected by centrifugation. Refractile inclusion bodies wereobserved in induced cultures indicating that the Fc-fusions were mostlikely produced in the insoluble fraction in E. coli. Cell pellets werelysed directly by resuspension in Laemmli sample buffer containing 10%β-mercaptoethanol and were analyzed by SDS-PAGE. In each case, anintense coomassie-stained band of the appropriate molecular weight wasobserved on an SDS-PAGE gel.

Purification of Fc-peptide fusion proteins. Cells are broken in water(1/10) by high pressure homogenization (2 passes at 14,000 PSI) andinclusion bodies are harvested by centrifugation (4200 RPM in J-6B for 1hour). Inclusion bodies are solubilized in 6M guanidine, 50 mM Tris, 8mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The solubilized mixture isdiluted 20 times into 2M urea, 50 mM tris, 160 mM arginine, 3 mMcysteine, pH 8.5. The mixture is stirred overnight in the cold and thenconcentrated about 10 fold by ultafiltration. It is then diluted 3 foldwith 10 mM Tris, 1.5M urea, pH 9. The pH of this mixture is thenadjusted to pH 5 with acetic acid. The precipitate is removed bycentrifugation and the supernatant is loaded onto a SP-Sepharose FastFlow column equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5 (10 mg/mlprotein load, room temperature). The protein is eluted from the columnusing a 20 column volume gradient in the same buffer ranging from 100 mMNaCl to 500 mM NaCl. The pool from the column is diluted 3 fold andloaded onto a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH 5(10mg/ml protein load, room temperature). The protein is eluted using a 20column volume gradient in the same buffer ranging from 150 mM NaCl to400 mM NaCl. The peak is pooled and filtered.

Characterization of activity of Fc-TNF-α inhibitor and TNF-αinhibitor-Fc. Binding of these peptide fusion proteins to TNF-α can becharacterized by BIAcore by methods available to one of ordinary skillin the art who is armed with the teachings of the present specification.

Example 5 IL-1 Antagonists

Fc-IL-1 antagonist. A DNA sequence coding for the Fc region of humanIgG1 fused in-frame to a monomer of an IL-1 antagonist peptide wasconstructed using standard PCR technology. The Fc and 5 glycine linkerportion of the molecule was generated in a PCR reaction with DNA fromthe Fc-EMP fusion strain #3718 (see Example 3) using the sense primer1216-52 and the antisense primer 2269-70 (SEQ ID NOS: 369 and 1118,respectively). The nucleotides encoding the IL-1 antagonist peptide wereprovided by the PCR primer 2269-70 shown below:

1216-52 AAC ATA AGT ACC TGT AGG ATC G 2269-70 CCG CGG ATC CAT TAC AGCGGC AGA GCG TAC GGC TGC CAG TAA CCC GGG GTC CAT TCG AAA CCA CCA CCT CCACCT TTA CCCThe oligonucleotide 2269-70 overlaps the glycine linker and Fc portionof the template by 22 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4506.

The nucleotide and amino acid sequences (SEQ ID NOS: 1059 and 1060) ofthe fusion protein are shown in FIGS. 21A and 21B.

IL-1 antagonist-Fc. A DNA sequence coding for an IL-1 antagonist peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. The template for the PCR reaction was a plasmidcontaining an unrelated peptide fused via a five glycine linker to Fc.The nucleotides encoding the IL-1 antagonist peptide were provided bythe sense PCR primer 2269-69, with primer 1200-54 serving as theantisense primer (SEQ ID NOS: 1119 and 407, respectively). The primersequences are shown below:

2269-69 GAA TAA CAT ATG TTC GAA TGG ACC CCG GGT TAC TGG CAG CCG TAC GCTCTG CCG CTG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT GCT CAG CGGTGG CAThe oligonucleotide 2269-69 overlaps the glycine linker and Fc portionof the template by 24 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4505.

The nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) ofthe fusion protein are shown in FIGS. 22A and 22B. Expression andpurification were carried out as in previous examples.

Characterization of Fc-IL-1 antagonist peptide and IL-1 antagonistpeptide-Fc activity. IL-1 Receptor Binding competition between IL-1β,IL-1RA and Fc-conjugated IL-1 peptide sequences was carried out usingthe IGEN system. Reactions contained 0.4 nM biotin-IL-1R+15 nMIL-1-TAG+3 uM competitor+20 ug/ml streptavidin-conjugate beads, wherecompetitors were IL-1RA, Fc-IL-1 antagonist, IL-1 antagonist-Fc).Competition was assayed over a range of competitor concentrations from 3uM to 1.5 pM. The results are shown in Table C below:

TABLE C Results from IL-1 Receptor Binding Competition Assay IL-1pep-FcFc-IL-1pep IL-1ra KI 281.5 59.58 1.405 EC50 530.0 112.2 2.645 95%Confidence Intervals EC50 280.2 to 1002 54.75 to 229.8 1.149 to 6.086 KI148.9 to 532.5 29.08 to 122.1 0.6106 to 3.233 Goodness of Fit R² 0.97900.9687 0.9602

Example 6 VEGF-Antagonists

Fc-VEGF Antagonist. A DNA sequence coding for the Fc region of humanIgG1 fused in-frame to a monomer of the VEGF mimetic peptide wasconstructed using standard PCR technology. The templates for the PCRreaction were the pFc-A3 plasmid and a synthetic VEGF mimetic peptidegene. The synthetic gene was assembled by annealing the following twooligonucleotides primer (SEQ ID NOS: 1110 and 1111, respectively):

2293-11 GTT GAA CCG AAC TGT GAC ATC CAT GTT ATG TGG GAA TGG GAA TGT TTTGAA CGT CTG 2293-12 CAG ACG TTC AAA ACA TTC CCA TTC CCA CAT AAC ATG GATGTC ACA GTT CGG TTC AAC

The two oligonucleotides anneal to form the following duplex encoding anamino acid sequence shown below (SEQ ID NOS; 1113 and 1114):

GTTGAACCGAACTGTGACATCCATGTTATGTGGGAATGGGAATGTTTTGAACGTCTG 1---------+---------+---------+---------+---------+------- 57CAACTTGGCTTGACACTGTAGGTACAATACACCCTTACCCTTACAAAACTTGCAGAC aV  E  P  N  C  D  I  H  V  M  W  E  W  E  C  F  E  R  L -This duplex was amplified in a PCR reaction using 2293-05 and 2293-06 asthe sense and antisense primers (SEQ ID NOS. 1122 and 1123).

The Fc portion of the molecule was generated in a PCR reaction with thepFc-A3 plasmid using the primers 2293-03 and 2293-04 as the sense andantisense primers (SEQ ID NOS. 1120 and 1121, respectively). The fulllength fusion gene was obtained from a third PCR reaction using theoutside primers 2293-03 and 2293-06. These primers are shown below:

2293-03 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GAC AAA ACT CAC ACA TGT2293-04 GTC ACA GTT CGG TTC AAC ACC ACC ACC ACC ACC TTT ACC CGG AGA CAGGGA 2293-05 TCC CTG TCT CCG GGT AAA GGT GGT GGT GGT GGT GTT GAA CCG AACTGT GAC ATC 2293-06 CCG CGG ATC CTC GAG TTA CAG ACG TTC AAA ACA TTC CCA

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4523.

The nucleotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) ofthe fusion protein are shown in FIGS. 23A and 23B.

VEGF antagonist-Fc. A DNA sequence coding for a VEGF mimetic peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. The templates for the PCR reaction were thepFc-A3 plasmid and the synthetic VEGF mimetic peptide gene describedabove. The synthetic duplex was amplified in a PCR reaction using2293-07 and 2293-08 as the sense and antisense primers (SEQ ID NOS. 1124and 1125, respectively).

The Fc portion of the molecule was generated in a PCR reaction with thepFc-A3 plasmid using the primers 2293-09 and 2293-10 as the sense andantisense primers (SEQ ID NOS. 1126 and 1127, respectively). The fulllength fusion gene was obtained from a third PCR reaction using theoutside primers 2293-07 and 2293-10. These primers are shown below:

2293-07 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GTT GAA CCG AAC TGT GAC2293-08 ACA TGT GTG AGT TTT GTC ACC ACC ACC ACC ACC CAG ACG TTC AAA ACATTC 2293-09 GAA TGT TTT GAA CGT CTG GGT GGT GGT GGT GGT GAC AAA ACT CACACA TGT 2293-10 CCG CGG ATC CTC GAG TTA TTT ACC CGG AGA CAG GGA GAG

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4524.

The nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) ofthe fusion protein are shown in FIGS. 24A and 24B. Expression andpurification were carried out as in previous examples.

Example 7 MMP Inhibitors

Fc-MMP inhibitor. A DNA sequence coding for the Fc region of human IgG1fused in-frame to a monomer of an MMP inhibitory peptide was constructedusing standard PCR technology. The Fc and 5 glycine linker portion ofthe molecule was generated in a PCR reaction with DNA from the Fc-TNF-αinhibitor fusion strain #4544 (see Example 4) using the sense primer1216-52 and the antisense primer 2308-67 (SEQ ID NOS: 369 and 1115,respectively). The nucleotides encoding the MMP inhibitor peptide wereprovided by the PCR primer 2308-67 shown below:

1216-52 AAC ATA AGT ACC TGT AGG ATC G 2308-67 CCG CGG ATC CAT TAG CACAGG GTG AAA CCC CAG TGG GTG GTG CAA CCA CCA CCT CCA CCT TTA CCCThe oligonucleotide 2308-67 overlaps the glycine linker and Fc portionof the template by 22 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4597.

The nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) ofthe fusion protein are shown in FIGS. 25A and 25B. Expression andpurification were carried out as in previous examples.

MMP Inhibitor-Fc. A DNA sequence coding for an MMP inhibitory peptidefused in-frame to the Fc region of human IgG1 was constructed usingstandard PCR technology. The Fc and 5 glycine linker portion of themolecule was generated in a PCR reaction with DNA from the Fc-TNF-αinhibitor fusion strain #4543 (see Example 4). The nucleotides encodingthe MMP inhibitory peptide were provided by the sense PCR primer2308-66, with primer 1200-54 serving as the antisense primer (SEQ IDNOS: 1116 and 407, respectively). The primer sequences are shown below:

2308-66 GAA TAA CAT ATG TGC ACC ACC CAC TGG GGT TTC ACC CTG TGC GGT GGAGGC GGT GGG GAC AAA 1200-54 GTT ATT GCT CAG CGG TGG CA

The two oligonucleotides anneal to form the following duplex encoding andamino acid sequence shown below (SEQ ID NOS: 1113 and 1114):

The oligonucleotide 2269-69 overlaps the glycine linker and Fc portionof the template by 24 nucleotides, with the PCR resulting in the twogenes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested withrestriction endonucleases NdeI and BamHI, and then ligated into thevector pAMG21 and transformed into competent E. coli strain 2596 cellsas described for EMP-Fc herein. Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedand designated Amgen strain #4598.

The nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) ofthe fusion protein are shown in FIGS. 26A and 26B.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto, without departing from the spirit and scope of theinvention as set forth herein.

Abbreviations

Abbreviations used throughout this specification are as defined below,unless otherwise defined in specific circumstances.

-   -   Ac acetyl (used to refer to acetylated residues)    -   AcBpa acetylated p-benzoyl-L-phenylalanine    -   ADCC antibody-dependent cellular cytotoxicity    -   Aib aminoisobutyric acid    -   bA beta-alanine    -   Bpa p-benzoyl-L-phenylalanine    -   BrAc bromoacetyl (BrCH₂C(O)    -   BSA Bovine serum albumin    -   Bzl Benzyl    -   Cap Caproic acid    -   CTL Cytotoxic T lymphocytes    -   CTLA4 Cytotoxic T lymphocyte antigen 4    -   DARC Duffy blood group antigen receptor    -   DCC Dicylcohexylcarbodiimide    -   Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl    -   EMP Erythropoietin-mimetic peptide    -   ESI-MS Electron spray ionization mass spectrometry    -   EPO Erythropoietin    -   Fmoc fluorenylmethoxycarbonyl    -   G-CSF Granulocyte colony stimulating factor    -   GH Growth hormone    -   HCT hematocrit    -   HGB hemoglobin    -   hGH Human growth hormone    -   HOBt 1-Hydroxybenzotriazole    -   HPLC high performance liquid chromatography    -   IL interleukin    -   IL-R interleukin-1 receptor    -   IL-1R interleukin-1 receptor    -   IL-1ra interleukin-1 receptor antagonist    -   Lau Lauric acid    -   LPS lipopolysaccharide    -   LYMPH lymphocytes    -   MALDI-MS Matrix-assisted laser desorption ionization mass        spectrometry    -   Me methyl    -   MeO methoxy    -   MHC major histocompatibility complex    -   MMP matrix metalloproteinase    -   MMPI matrix metalloproteinase inhibitor    -   1-Nap 1-napthylalanine    -   NEUT neutrophils    -   NGF nerve growth factor    -   Nle norleucine    -   NMP N-methyl-2-pyrrolidinone    -   PAGE polyacrylamide gel electrophoresis    -   PBS Phosphate-buffered saline    -   Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl    -   PCR polymerase chain reaction    -   Pec pipecolic acid    -   PEG Poly(ethylene glycol)    -   pGlu pyroglutamic acid    -   Pic picolinic acid    -   PLT platelets    -   pY phosphotyrosine    -   RBC red blood cells    -   RBS ribosome binding site    -   RT room temperature (25° C.)    -   Sar sarcosine    -   SDS sodium dodecyl sulfate    -   STK serine-threonine kinases    -   t-Boc tert-butoxycarbonyl    -   tBu tert-butyl    -   TGF tissue growth factor    -   THF thymic humoral factor    -   TK tyrosine kinase    -   TMP Thrombopoietin-mimetic peptide    -   TNF Tissue necrosis factor    -   TPO Thrombopoietin    -   TRAIL TNF-related apoptosis-inducing ligand    -   Trt trityl    -   UK urokinase    -   UKR urokinase receptor    -   VGEF vascular endothelial cell growth factor    -   VIP vasoactive intestinal peptide    -   WBC white blood cells

1. A composition of matter of the formula(X¹)_(a)—F¹—(X²)_(b) and multimers thereof, wherein: F¹ is an Fc domain;X¹ and X² are each independently selected from -(L¹)_(c)-P¹,-(L¹)_(c)-P¹-(L²)_(d)-P², -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴ P¹, P², P³, and P⁴ areeach independently randomized TNF-α binding peptide sequences; L¹, L²,L³, and L⁴ are each independently linkers; and a, b, c, d, e, and f areeach independently 0 or 1, provided that at least one of a and b is 1and wherein “peptide” refers to molecules 2 to 40 amino acid and whereinneither X¹ nor X² is a native protein.
 2. The composition of matter ofclaim 1 of the formulaeX¹—F¹orF¹—X².
 3. The composition of matter of claim 1 of the formulaF¹-(L¹)_(c)-P¹.
 4. The composition of matter of claim 1 of the formulaF¹-(L¹)^(c)-P¹-(L²)^(d)-P².
 5. The composition of matter of claim 1wherein F₁ is an IgG Fc domain.
 6. The composition of matter of claim 1wherein F¹ is an IgG1 Fc domain.
 7. The composition of matter of claim 1wherein F¹ comprises the sequence of SEQ ID NO:
 2. 8. The composition ofmatter of claim 1 wherein the TNF-α binding peptide sequences areselected from SEQ ID NOS: 106 to 123 and
 1085. 9. The composition ofmatter of claim 1, wherein the TNF-α binding peptide sequences areselected from Table
 8. 10. The composition of matter of claim 1comprising a sequence selected from SEQ ID NOS: 1045 and 1046.